Chlorine dioxide treatment for biological tissue

ABSTRACT

Methods, compositions, devices, and systems for administration to a biological tissue of a composition comprising a chlorine dioxide source are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit pursuant to 35 U.S.C. §119(e) ofU.S. Provisional Application Nos. U.S. Provisional Application Nos.61/149,784, filed Feb. 4, 2009; 61/150,685, filed Feb. 6, 2009; and61/187,198, filed Jun. 15, 2009, each of which is hereby incorporatedherein by reference in their entirety.

BACKGROUND

Chlorine dioxide is known to be a disinfectant, as well as a strongoxidizing agent. The bactericidal, algaecidal, fungicidal, bleaching,and deodorizing properties of chlorine dioxide are also well known.Therapeutic and cosmetic applications for chlorine dioxide are known.For example, U.S. Pat. No. 6,280,716 describes the use of stabilizedchlorine dioxide solutions for the treatment of vaginal itching. U.S.Pat. No. 7,029,705 describes the use of stabilized chlorine dioxidesolutions for a method of nasal hygiene.

The traditional method for preparing chlorine dioxide involves reactingsodium chlorite with gaseous chlorine (Cl₂(g)), hypochlorous acid(HOCl), or hydrochloric acid (HCl). The reactions proceed at muchgreater rates in acidic medium, so substantially all traditionalchlorine dioxide generation chemistry results in an acidic productsolution having a pH below 3.5.

Chlorine dioxide may also be prepared from chlorate anion by eitheracidification or a combination of acidification and reduction. Atambient conditions, all reactions using chlorate anion require stronglyacidic conditions; most commonly in the range of 7-9 N. Heating of thereagents to higher temperature and continuous removal of chlorinedioxide from the product solution can reduce the acidity needed to lessthan 1 N.

A method of preparing chlorine dioxide in situ uses a solution referredto as “stabilized chlorine dioxide.” Stabilized chlorine dioxidesolutions contain little or no chlorine dioxide, but rather, consistsubstantially of sodium chlorite at neutral or slightly alkaline pH.Addition of an acid to the sodium chlorite solution activates the sodiumchlorite, and chlorine dioxide is generated in situ in the solution. Theresulting solution is acidic. Typically, the extent of sodium chloriteconversion to chlorine dioxide is low, and a substantial quantity ofsodium chlorite remains in the solution.

The current literature summarized above describes the use of chlorinedioxide compositions and methods that are damaging to biologicaltissues. Methods, compositions, devices, and systems for using chlorinedioxide for treatment of biological tissue in which biological tissue isnot damaged are needed.

SUMMARY

The following embodiments meet and address these needs. The followingsummary is not an extensive overview. It is intended to neither identifykey or critical elements of the various embodiments, not delineate thescope of them.

Provided is a method for treating a wound in a tissue comprising thestep of administering to the wound a composition comprising a chlorinedioxide source to provide an efficacious amount of chlorine dioxide tothe wound, thereby treating the wound. The administering step comprisesone or more of: i) contacting the wound with a substantiallynon-cytotoxic and/or substantially non-irritating composition comprisingthe chlorine dioxide source; ii) contacting the wound with a devicecomprising the chlorine dioxide source and oxy-chlorine anions, whereinthe device delivers a substantially oxy-chlorine anion free chlorinedioxide composition to the tissue; or iii) contacting the wound with acomposition comprising the chlorine dioxide source and oxy-chlorineanions; and a barrier substance that substantially prohibits passagetherethrough of the oxy-chlorine anions and permits passage therethroughof a substantially oxy-chlorine anion free chlorine dioxide composition,thereby enabling delivery of the substantially oxy-chlorine anion freechlorine dioxide composition to the wound.

In an embodiment, the method further comprises a second iteration of thecontacting step, wherein the second iteration is substantiallycontiguous with the first iteration. In another embodiment, the methodfurther comprises at least a third iteration of the contacting step,wherein the third iteration is substantially contiguous with the seconditeration and the second iteration is substantially contiguous with thefirst iteration.

In some embodiments, the method comprises contacting the wound with asubstantially non-cytotoxic and/or substantially non-irritatingcomposition comprising the chlorine dioxide source to form acomposition-contacted wound, and further comprising applying ultrasonicenergy to the composition-contacted wound. In other embodiments, theadministration step comprises contacting the wound with a devicecomprising the chlorine dioxide source and oxy-chlorine anions to form adevice-contacted wound, and further comprising applying ultrasonicenergy to the device-contacted wound. In certain embodiments, theultrasonic energy frequency is between about 10 kHz to about 100 kHzwith a power intensity of about 2 W/cm² to about 3.5 W/cm².

In other embodiments, the method comprises irrigating the wound with asubstantially non-cytotoxic and/or substantially non-irritatingcomposition using an irrigation device. In some embodiments, asubstantially non-cytotoxic and/or substantially non-irritatingcomposition comprises less than about 0.2 milligrams oxy-chlorine anionper gram composition. In yet other embodiments, the administration stepcomprises contacting the wound with a device comprising the chlorinedioxide source and oxy-chlorine anions, wherein the device is anirrigation device that delivers a substantially oxy-chlorine anion freechlorine dioxide composition to the wound.

In certain embodiments, the administration step comprises contacting thewound with a composition comprising the chlorine dioxide source andoxy-chlorine anions; and a barrier substance that substantiallyprohibits passage therethrough of the oxy-chlorine anions and permitspassage therethrough of a substantially oxy-chlorine anion free chlorinedioxide composition, thereby enabling delivery of the substantiallyoxy-chlorine anion free chlorine dioxide composition to the wound toform a composition-contacted wound, and further comprising applyingultrasonic energy to the composition-contacted wound.

In some embodiments of the method of treating a wound in a tissue, thecomposition comprises about 1 to about 1000 ppm chlorine dioxide. Insome embodiments, an actual dosage of at least about 200 ppm-minutes ofchlorine dioxide is administered. In some embodiments, the chlorinedioxide source comprises chlorine-dioxide generating components, whereinsaid chlorine-dioxide generating components are particulate precursor ofchlorine dioxide. Optionally, in any of the embodiments, the compositionfurther comprises a second therapuetic agent. In other embodiments, themethod further comprises administering a second composition comprising asecond therapuetic agent to the wound.

In some embodiments, the method of treating a wound in a tissuecomprises contacting the wound with a substantially non-cytotoxic and/orsubstantially non-irritating composition comprising the chlorine dioxidesource, the composition comprising less than about 0.2 milligramsoxy-chlorine anion per gram composition. In other embodiments, themethod of treating a wound in a tissue comprises the compositioncomprises a pH from about 4.5 to about 11.

Further provided is a system for irrigating a biological tissue, thesystem comprising: a device and a source of a fluid comprising chlorinedioxide. The irrigation device comprises: 1) a flexible, semi-rigid orrigid pouch or other containment chamber with inlet and outlet ports andwith an opening to contact at least a portion of a tissue targeted to beirrigated; 2) a fluid supply and egress system connected to the chamberports so as to provide fluid to or drain fluid from the chamber; 3) ameans of maintaining contact of the chamber to the tissue whichsurrounds the target tissue so as to form a tight substantiallyleak-proof seal; 4) an optional open cell foam or other porous materialplaced inside the pouch to ensure a uniform distribution of flow; and 5)a fluid handling unit which supplies fluid to and/or allows fluid toexit the containment chamber, wherein the fluid handling unit suppliesfluid from the source of the fluid comprising chlorine dioxide.

A method for alleviating an oral cavity tissue infection is alsoprovided. The method comprises administering to infected tissue in anoral cavity a composition comprising a chlorine dioxide source toprovide an efficacious amount of chlorine dioxide to the tissue, whereinthe administering step comprises one or more of: i) contacting thetissue with a substantially non-cytotoxic and/or substantiallynon-irritating composition comprising the chlorine dioxide source toform a composition-contacted oral tissue, and applying ultrasonic energyto the composition-contacted oral tissue; ii) contacting the tissueiteratively with at least two, substantially contiguous applications ofa substantially non-cytotoxic composition comprising the chlorinedioxide source; iii) irrigating the tissue with a substantiallynon-cytotoxic composition comprising the chlorine dioxide source usingan irrigation device; or

iv) irrigating the tissue using an irrigation device that delivers asubstantially oxy-chlorine anion free chlorine dioxide composition tothe wound.

In some embodiments, the composition comprising the chlorine dioxidesource comprises about 1 to about 1000 ppm chlorine dioxide. In someembodiments, the composition comprising the chlorine dioxide source hasa pH from about 4.5 to about 11. In some embodiments, the chlorinedioxide source comprises chlorine-dioxide generating components, whereinsaid chlorine-dioxide generating components are particulate precursor ofchlorine dioxide. In some embodiments, the substantially non-cytotoxicand/or substantially non-irritating composition comprises less thanabout 0.2 milligrams oxy-chlorine anion per gram composition.Optionally, in any of the embodiments, the composition further comprisesa second therapuetic agent. In other embodiments, the method furthercomprises administering a second composition comprising a secondtherapuetic agent to the infected oral cavity tissue.

In addition, a method of whitening a tooth surface is provided. Themethod comprises contacting a surface of a tooth with an efficaciousamount of a composition comprising a chlorine dioxide source to providean efficacious amount of chlorine dioxide to the tooth surface, whereinthe contacting step comprises one or more of: i) contacting the toothsurface iteratively with at least two, substantially contiguousapplications of a substantially non-cytotoxic and/or substantiallynon-irritating composition comprising a chlorine dioxide source; ii)irrigating the tooth surface using an irrigation device that delivers asubstantially non-cytotoxic and/or substantially non-irritatingcomposition comprising a chlorine dioxide source, or iii) irrigating thetooth surface using an irrigation device that delivers a substantiallyoxy-chlorine anion free chlorine dioxide composition to the toothsurface.

In some embodiments, the composition comprising the chlorine dioxidesource comprises about 1 to about 1000 ppm chlorine dioxide. In someembodiments, the composition comprising the chlorine dioxide source hasa pH from about 4.5 to about 11. In some embodiments, the chlorinedioxide source comprises chlorine-dioxide generating components, whereinsaid chlorine-dioxide generating components are particulate precursor ofchlorine dioxide. In some embodiments wherein the contacting stepcomprises contacting the tooth surface iteratively with at least two,substantially contiguous applications of a substantially non-cytotoxicand/or substantially non-irritating composition comprising a chlorinedioxide source, each application comprises a chlorine dioxide dosage ofabout 750 ppm-minutes to about 2000 ppm-minutes. In some embodiments, atleast four substantially contiguous applications are administered. Insome embodiments, the substantially non-cytotoxic and/or substantiallynon-irritating composition comprises less than about 0.2 milligramsoxy-chlorine anion per gram composition.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the various compositions and methods,there are depicted in the drawings certain embodiments of the methods,compositions, and devices disclosed. However, the compositions and theirmethods of use are not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

FIG. 1 is a graph depicting the reduction in log of bacteria (log kill)in various biofilms as a function of chlorine dioxide (ClO₂) dosage.MRSA=methicillin resistance Staphylococcus aureus. PA=Pseudomonasaeruginosa.

FIG. 2 is a graph depicting representative data for decrease in chlorinedioxide (ClO₂) as a function of time in a protein-rich environment. Thedata depicted were measured for a solution containing 6.29% by wt. fetalblood serum (FBS) and having an initial weight ratio (mg/mg) of FBS/ClO₂of nominally 500.

FIG. 3 is a graph of chlorine dioxide decay rates as a function of theinitial weight (mg/mg) ratio of fetal bovine serum (FBS) to chlorinedioxide.

FIG. 4 is a graph depicting the reduction in log bacteria in variousbiofilms as a function of corrected chlorine dioxide dosage. Chlorinedioxide dosage was corrected to reflect the chlorine dioxide consumptionattributable to reaction with organic material (e.g., proteins) otherthan the bacteria.

FIG. 5 is a schematic representation of the wound placement in anassessment of various delivery methods for contacting a wound withchlorine dioxide. D-2 indicates that the wound was biopsied on Day 2.

FIG. 6 is a bar graph depicting the total bacteria log in wounds afterdifferent delivery methods. The y-axis is log total bacteria incolony-forming units (CFUs) per gram tissue. Moist control: data forwounds A1, B1, and D1. Irrigation pump: data for wounds A2-A4. NaCMCgel: data for wounds B2-B4. HPMC gel: data for wounds C2-C4. Irrigationsyringe: data for wounds D2-D4. Manual irrigation: data for wound C1.NaCMC=sodium carboxymethylcellulose. HPMC=hydroxypropyl methylcellulose.

FIG. 7 is a bar graph depicting the total log of coagulase-negativeStaphylococci in wounds after different delivery methods. The y-axis islog coagulase-negative Staph in CFUs per gram tissue. Moist control:data for wounds A1, B1, and D1. Irrigation pump: data for wounds A2-A4.NaCMC gel: data for wounds B2-B4. HPMC gel: data for wounds C2-C4.Irrigation syringe: data for wounds D2-D4. Manual irrigation: data forwound C1.

FIG. 7 is a bar graph depicting the total log of coagulase-negativeStaph in wounds after different delivery methods. The y-axis is logcoagulase-negative Staph in (colony-forming units) per gram tissue.Moist control: data for wounds A1, B1, and D1. Irrigation pump: data forwounds A2-A4. NaCMC gel: data for wounds B2-B4. HPMC gel: data forwounds C2-C4. Irrigation syringe: data for wounds D2-D4. Manualirrigation: data for wound C1.

FIG. 8 is a bar graph depicting the total log of Pseudomonas in woundsafter different delivery methods. The y-axis is log Pseudomonas in CFUsper gram tissue. Moist control: data for wounds A1, B1, and D1.Irrigation pump: data for wounds A2-A4. NaCMC gel: data for woundsB2-B4. HPMC gel: data for wounds C2-C4. Irrigation syringe: data forwounds D2-D4. Manual irrigation: data for wound C1.

DETAILED DESCRIPTION

Provided herein are methods of delivering a chlorine-dioxide containingcomposition to a tissue or biological material. The methods are usefulin the treatment of wounds, alleviation of an oral tissue infection andin tooth whitening.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art. Generally, the nomenclature used herein andthe laboratory procedures in cytopathicity analysis, microbial analysis,organic and inorganic chemistry, and dental clinical research are thosewell known and commonly employed in the art.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it can be used.Generally, “about” encompasses a range of values that are plus/minus 10%of a reference value. For instance, “about 25%” encompasses values from22.5% to 27.5%.

It is understood that any and all whole or partial integers between anyranges set forth herein are included herein.

As used herein, “biocidal” refers to the property of inactivating orkilling pathogens, such as bacteria, algae, and fungi.

As used herein, “tooth whitening” refers to a lightening of tooth shaderelative to the tooth shade prior to treatment. Lightening can beassessed on an isolated or an in situ tooth by standard, art-recognizedmethods of assessing tooth shade, which include qualitative,quantitative and semi-quantitative methods. For instance, lightening maybe assessed by simple visual inspection, e.g., by comparing “before” and“after” photographs of the treated teeth. Alternatively, a tooth may bedeemed whitened when the tooth shade relative to the tooth shade priorto treatment is two or more shades lighter, as assessed by Vitaclassical shade guide (typically under controlled visible lightconditions) or two or more levels as assessed using the VitaBleachedguide 3D-MASTER Shade system, which utilizes a multiple colorspectrophotometer and includes half lightness levels. A difference ofone shade is referred to herein as a “shade value unit” (SVU). Thus, forexample, a difference of two shades is a 2 SVU difference.

“Bleaching agent” as used herein refers to the active ingredient, orcombination of ingredients, in a composition that causes the lighteningand/or removal of the chromagens that contribute to the dark shade of atooth.

As used herein, a “biofilm” refers to a biological aggregate that formsa layer on a surface, the aggregate comprising a community ofmicroorganisms embedded in an extracellular matrix of polymers.Typically, a biofilm comprises a diverse community of microorganisms,including bacteria (aerobic and anaerobic), algae, protozoa, and fungi.Monospecies biofilms also exist.

As used herein, an “efficacious amount” of an agent is intended to meanany amount of the agent that will result in a desired biocidal effect, adesired cosmetic effect, and/or a desired therapeutic biological effect.In one example, an efficacious amount of an agent used for toothwhitening is an amount that will result in whitening of a tooth with oneor more treatments. In another example, an efficacious amount of anagent used for wound treatment is an amount that will result in astatistically significant improvement in wound healing such as areduction in bacterial level in the wound.

As used herein, a “wound” refers to a laceration, abrasion, puncture,burn, and/or other injury to any one or more soft and/or hard tissue.Exemplary tissues considered for such wound treatment include mucosaltissue and dermal tissue including epidermal tissue, dermal tissue, andsubcutaneous tissue (also called hypodermis tissue). As used herein, awound also encompasses a laceration, a puncture, and/or an avulsion of afingernail or toenail. A wound can penetrate the tissue partially orcompletely. A wound can arise accidently or intentionally, e.g., asurgical wound.

As used herein, a wound is “treated” if one or more indications of woundhealing are improved to a statistically significant amount or degree.Indications of wound healing are well known in the art. Indications ofwound healing can be present in any one or more of the four generaloverlapping phases of wound healing: hemostasis phase, inflammatoryphase, proliferative phase, and remodeling phase, and include the extentor the duration of each phase or of the overall healing process. Otherexamples of indications of wound healing include reduction in totalbacterial count, reduction in bacterial count of a specific bacteriasuch as Pseudomonas aeruginosa or Staphylococci, reduction in extent orduration of inflammation, increase in extent of or rate of woundcontraction, reduction in overall time to healing, and the like.

As used herein, “biological tissue” refers to soft biological tissue andhard biological tissue.

As used herein, a “soft biological tissue” refers to mucosal tissue anddermal tissue. Dermal tissue encompasses epidermal tissue, dermaltissue, and subcutaneous tissue (also called hypodermis tissue). “Softtissue” is used herein interchangeably with “soft biological tissue.”

As used herein, “hard biological tissue” refers to toe and finger nails,hard keratinized tissues, hard tooth tissue, and the like, found inanimals such as mammals. “Hard tissue” is used herein interchangeablywith “hard biological tissue.”

As used herein, “hard tooth tissue” refers to at least one of enamel anddentin.

As used herein, “hard tooth tissue damage” refers to at least one of areduction of microhardness of enamel, a reduction of microhardness ofdentin, an increase in the surface roughness of enamel and an increasein the surface roughness of dentin.

As used herein, a composition “does not substantially damage hard toothtissue” if one or more of the following is met for a tooth aftertreatment relative to the tooth prior to treatment: 1) enamelmicrohardness is decreased by an amount less than about 15% and/or thereduction is not statistically significant at the 5% confidence level;2) dentin microhardness is decreased by an amount less than about 15%and/or the reduction is not statistically significant at the 5%confidence level; 3) enamel surface roughness is increased by an amountno more than about 20% and/or the increase is not statisticallysignificant at the 5% confidence level; and 4) dentin surface roughnessis increased by an amount no more than about 8% and/or the increase isnot statistically significant at the 5% confidence level.

As used herein, “remineralization” refers to the process of repair ofacid damaged tooth structure by the recrystallization of mineral saltson or within the tooth architecture.

As used herein, “demineralization” refers to the process of mineral lossfrom teeth caused by acid, chelating agents or other accelerants ofdissolution. Demineralization can occur on tooth surfaces and/or belowtooth surfaces, depending on the composition of the demineralizingagent, the contacting medium, the concentration, and the pH.

As used herein, “irrigation” of a tissue refers to rinsing the tissuewith a solution. “Continuous irrigation” refers to rinsing the tissuewith a substantially steady stream of the solution for the duration ofthe treatment. “Intermittent irrigation” refers to rinsing the tissuewith a stream of fluid that is periodically slowed or stopped during thetreatment.

As used herein, a “plurality” refers to two or more. For instance, atreatment having a plurality of contacting steps encompasses two, three,four, five, six, seven, eight or more steps of contacting.

As used herein, a “chlorine dioxide source” refers to one of chlorinedioxide, chlorine dioxide-generating components, or a combination ofthereof.

As used herein, an “oral cavity infection” refers to a disease ordisorder of a tissue in an oral cavity caused by a pathogenic infection.The pathogen may be bacterial, viral, or fungal. An oral diseaseencompasses conditions wherein if the disease is not ameliorated thenthe animal's oral health continues to deteriorate. In contrast, an oraldisorder is a state of oral health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of oral health. The term encompasses periodontaldisease, halitosis, thrush, and dental caries development.

As used herein, a “periodontal disease” is an infection of the tissuesthat support a subject's teeth, caused by a pathogenic infection.Periodontal disease includes gingivitis and periodontitis.

As used herein, “dental plaque” refers to a biofilm that forms on thesurface of teeth.

As used herein, a disease or disorder is “alleviated” if the severity ofa symptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

As used herein, “cytotoxic” refers to the property of causing lethaldamage to mammalian cell structure or function. A composition is deemed“substantially non-cytotoxic” or “not substantially cytotoxic” if thecomposition meets the United States Pharmacopeia (USP) biologicalreactivity limits of the Agar Diffusion Test of USP <87> “BiologicalReactivity, in vitro,” (approved protocol current in 2007) when theactive pharmaceutical ingredient (API) is present in an efficaciousamount.

As used herein, “irritating” refers to the property of causing a localinflammatory response, such as reddening, swelling, itching, burning, orblistering, by immediate, prolonged, or repeated contact. For example,inflammation of a non-oral mucosal or dermal tissue in a mammal can bean indication of irritation to that tissue. A composition is deemed“substantially non-irritating” or “not substantially irritating,” if thecomposition is judged to be slightly or not irritating using anystandard method for assessing dermal or mucosal irritation. Non-limitingexamples of methods useful for assessing dermal irritation include theuse of in vitro tests using tissue-engineered dermal tissue, such asEpiDerm™ (MatTek Corp., Ashland, Mass.), which is a human skin tissuemodel (see, for instance, Chatterjee et al., 2006, Toxicol Letters 167:85-94) or ex vivo dermis samples. Non-limiting examples of methodsuseful for mucosal irritation include: HET-CAM (hen's eggtest-chorioallantoic membrane); slug mucosal irritation test; and invitro tests using tissue-engineered nasal or sinus mucosa orvaginal-ectocervical tissues. Other useful methods of irritationmeasurement include in vivo methods, such as dermal irritation of rat orrabbit skin (e.g., the Draize skin test (OECD, 2002, Test Guidelines404, Acute Dermal Irritation/Corrosion) and EPA Health Effects TestingGuidelines; OPPTS 870.2500 Acute Dermal Irritation). The skilled artisanis familiar with art-recognized methods of assessing dermal and mucosalirritation.

As used herein, “oxy-chlorine anion” refers to chlorite (ClO₂) and/orchlorate (ClO₃ ⁻) anions.

As used herein, “substantially pure chlorine dioxide solution” refers toa solution of chlorine dioxide that has a non-cytotoxic concentration ofoxy-chlorine anion. As used herein, “substantially pure chlorine dioxidesolution” also refers to a concentrated solution of chlorine dioxidethat contains a concentration of oxy-chlorine anion that upon dilutionto an efficacious amount of chlorine dioxide is not cytotoxic withrespect to the concentration of oxy-chlorine anion.

By “substantially oxy-chlorine anion free chlorine dioxide composition”is meant a composition that contains an efficacious amount of chlorinedioxide and a non-cytotoxic and/or non-irritating concentration ofoxychlorine anion, all as defined hereinabove. The composition maycontain other components or may consist essentially of oxy-chlorineanion free chlorine dioxide. The composition may be a gas or vaporcomprising or consisting essentially of chlorine dioxide, but may be anytype of fluid, including a solution or a thickened fluid. Thecomposition may be an aqueous fluid or a non-aqueous fluid.

By “stable” is meant that the components used to form chlorine dioxide,i.e., the chlorine dioxide forming ingredients, are not immediatelyreactive with each other to form chlorine dioxide. It will be understoodthat the components may be combined in any fashion, such as sequentiallyand/or simultaneously, so long as the combination is stable until suchtime that ClO₂ is to be generated.

By “non-reactive” is meant that a component or ingredient as used is notimmediately reactive to an unacceptable degree with other components oringredients present to form chlorine dioxide or mitigate the ability ofany component or ingredient to perform its function in the formulationat the necessary time. As the skilled artisan will recognize, theacceptable timeframe for non-reactivity will depend upon a number offactors, including how the formulation is to be formulated and stored,how long it is to be stored, and how the formulation is to be used.Accordingly, “not immediately reactive” will range from one or moreminutes, to one or more hours, to one or more weeks.

The phrase “thickened fluid composition” encompasses compositions whichcan flow under applied shear stress and which have an apparent viscositywhen flowing that is greater than the viscosity of the correspondingaqueous chlorine dioxide solution of the same concentration. Thisencompasses the full spectrum of thickened fluid compositions,including: fluids that exhibit Newtonian flow (where the ratio of shearrate to shear stress is constant and viscosity is independent of shearstress), thixotropic fluids (which require a minimum yield stress to beovercome prior to flow, and which also exhibit shear thinning withsustained shear), pseudoplastic and plastic fluids (which require aminimum yield stress to be overcome prior to flow), dilantant fluidcompositions (which increase in apparent viscosity with increasing shearrate), and other materials which can flow under applied yield stress.

A “thickener component,” as the phrase is used herein, refers to acomponent that has the property of thickening a solution or mixture towhich it is added. A “thickener component” can be used to make a“thickened fluid composition” as described above.

By “apparent viscosity” is meant the ratio of shear stress to shear rateat any set of shear conditions that result in flow. Apparent viscosityis independent of shear stress for Newtonian fluids and varies withshear rate for non-Newtonian fluid compositions.

The term “particulate” is defined to mean all solid materials. By way ofa non-limiting example, particulates may be interspersed with each otherto contact one another in some way. These solid materials includeparticles comprising big particles, small particles or a combination ofboth big and small particles.

As used herein, “NaDCCA” refers to sodium dichloroisocyanurate and/orthe dihydrate thereof.

By “source of free halogen” and “free halogen source” is meant acompound or mixtures of compounds which release halogen upon reactionwith water.

By “free halogen” is meant halogen as released by a free halogen source.

By “particulate precursor of chlorine dioxide” is meant a mixture ofchlorine-dioxide-forming components that are particulate. Granules ofASEPTROL (BASF, Florham Park, N.J.) are an exemplary particulateprecursor of chlorine dioxide.

By “solid body” is meant a solid shape, typically a porous solid shape,or a tablet comprising a mixture of granular particulate ingredientswherein the size of the particulate ingredients is substantially smallerthan the size of the solid body; by “substantially smaller” is meant atleast 50% of the particles have a particle size at least one order ofmagnitude, and preferably at least two orders of magnitude, smaller thanthe size of solid body.

The term “hydrophobic” or “water-insoluble,” as used with respect toorganic polymers refers to an organic polymer, which has a watersolubility of less than about one gram per 100 grams of water at 25° C.

By “acid source” is meant a material, usually a particulate solidmaterial, which is itself acidic or produces an acidic environment whenin contact with liquid water or solid oxy-chlorine anion.

A “matrix,” as used herein, is a material that functions as a protectivecarrier of chlorine dioxide-generating components. A matrix is typicallya continuous solid or fluid phase in which the materials that canparticipate in a reaction to form chlorine dioxide are suspended orotherwise contained. The matrix can provide physical shape for thematerial. If sufficiently hydrophobic, a matrix may protect thematerials within from contact with moisture. If sufficiently rigid, amatrix may be formed into a structural member. If sufficiently fluid, amatrix may function as a vehicle to transport the material within thematrix. If sufficiently adhesive, the matrix can provide a means toadhere the material to an inclined or vertical, or horizontal downwardsurface. A fluid matrix may be a liquid such that it flows immediatelyupon application of a shear stress, or it may require that a yieldstress threshold be exceeded to cause flow. In some embodiments, thematrix can be either a fluid, or capable of becoming fluid (e.g., uponheating) such that other components may be combined with and into thematrix (e.g., to initiate reaction to form chlorine dioxide). In otherembodiments, the matrix is a continuous solid; chlorine dioxidegeneration can be initiated by, for instance, penetration of water orwater vapor, or by light activation of an energy-activatable catalyst.

By “film” is meant a layer of a material having two dimensionssubstantially larger than the third dimension. A film may be a liquid ora solid material. For some materials, a liquid film can be convertedinto a solid film by curing, for instance, by evaporation, heating,drying and/or cross-linking.

Unless otherwise indicated or evident from context, preferencesindicated above and herein apply to the entirety of the embodimentsdiscussed herein.

Description

Disclosed are methods of delivering chlorine dioxide to a tissue, suchas a wound, infected oral tissue, or hard biological tissue such as atooth surface. The methods can be practiced on the tissue of any animal.Non-limiting examples of animals are mammals, such as humans, non-humanprimates, domesticated animals such as cattle, horses, dogs, sheep,goats, and pigs, and rodents such as mice and rats. In an embodiment,the methods are practiced on a human tissue.

Chlorine dioxide has well-documented potent biocidal activity.Disadvantageously, chlorine dioxide-containing compositions of the priorart can be cytotoxic and irritating to soft tissues such as mucosaltissue or dermal tissue, and damaging to hard tissues such as hard toothtissue. The cytotoxicity of chlorine dioxide-containing compositionsresults predominantly from the presence of oxy-chlorine anions, and notfrom the presence of chlorine, which can be a product of chlorinedioxide decomposition. By substantially preventing or inhibitingoxy-chlorine anions present in a chlorine-dioxide containing compositionfrom contacting cells and tissues, including hard tooth tissues such asenamel and dentin and soft tissues, such as wound tissue or oral mucosaand gums, that are targeted for treatment, tissue damage can bemeasurably reduced or minimized, while achieving the biocidal efficacyor bleaching efficacy of chlorine dioxide.

As shown herein, chlorine dioxide is efficacious in disrupting,penetrating and/or otherwise inactivating biofilms on surfaces,including biofilms of methicillin resistant Staphylococcus aureus(MRSA), Pseudomonas aeruginosa (PA) or a combination thereof. MRSA is aresistant variation of the common bacterium Staphylococcus aureus. Ithas evolved an ability to survive treatment withbeta-lactamase-resistant beta-lactam antibiotics, including methicillin,dicloxacillin, nafcillin, and oxacillin. PA is an opportunistic pathogenwith a low intrinsic antibiotic sensitivity and the capacity to readilyacquire antibiotic resistance. PA has emerged as a nosocomial pathogenof increasing clinical relevance. It is demonstrated herein thatchlorine dioxide is efficacious in disrupting, penetrating, and/orotherwise inactivating biofilms, even in the presence of a protein-richenvironment. As further shown herein, administration of chlorine dioxideto a wound in accordance with the methods described can reduce totalbacterial log count in the wound. It is, further demonstrated that onecan estimate the effect of blood serum in a wound on the rate ofconsumption of chlorine dioxide, therefore one can estimate dosage (inppm-min) for efficacy with more accuracy. It is also shown herein thataltering the specific treatment regimen of treating a tooth surface witha chlorine dioxide composition can affect the extent of tooth whiteningfor a given dosage (in ppm-min) to a statistically significant amount.

Accordingly, the methods described herein generally pertain to theadministration of a composition comprising a chlorine dioxide source toa wound in a substantially non-cytotoxic and/or non-irritating manner totreat the wound.

A wound refers to a laceration, abrasion, puncture or other injury toone or more tissues. The tissue can be any of mucosal tissue, dermaltissue including epidermal tissue, dermal tissue, and subcutaneoustissue (also called hypodermis tissue), and hard tissue such asfingernail and toenail. A wound can penetrate the tissue partially orcompletely. A wound can arise via accidental trauma or intentionally,e.g., a surgical wound. Wounds range from acute to chronic. Acute woundsheal in an orderly set of stages and in a relatively sort period oftime. Chronic wounds are wounds of long duration, e.g., greater than 3months, that heal very slowly. The most common chronic wounds are venousulcers, diabetic ulcers and pressure ulcers. Bacterial colonization is aproblem in both acute wounds and chronic wounds. The immune response tobacterial colonization can prolong wound inflammation, delay healing andcause tissue damage. Bacterial colonization characterized by thepresence of biofilm is particularly difficult to treat adequately withthe therapetics currently available. As shown herein, chlorine dioxideis a robust agent for disrupting, penetrating and/or otherwiseinactivating biofilm, even in the presence of a protein-richenvironments such as the exudate present in a wound bed.

In some embodiments, the method of treating a wound comprises contactingthe tissue with a substantially non-cytotoxic and/or non-irritatingcomposition. In other embodiments, the method comprises contacting thewound with a device or composition that delivers a substantiallyoxy-chlorine anion free chlorine dioxide composition to the wound. Inyet other embodiments, contact comprises irrigation of the wound tissue.In some embodiments, a combination therapy is carried out, such asincluding a step of improving tissue penetration of chlorine dioxide,for instance by use of ultrasonic energy.

The methods described herein also generally pertain to theadministration of a composition comprising chlorine dioxide to a tissuein a substantially non-cytotoxic and/or non-irritating manner toalleviate an infection of an oral tissue.

The methods described herein are useful in the treatment of anyinfection of any oral cavity tissue susceptible to topical exposure of abiocidal agent, in particular, chlorine dioxide. Infections of oralcavity tissue include, but are not limited to, halitosis, gingivitis,periodontitis, caries formation, and thrush. Oral tissue may be intactor may have one or more incisions, lacerations or othertissue-penetrating opening. The methods may be practicedprophylactically or therapeutically.

Bacteria in the oral cavity can produce volatile sulfur compounds (VSCs)which underlie oral malodor or halitosis. VSCs include hydrogen sulfide,methylmercaptan and dimethylmercaptan. Exemplary bacteria that cancontribute to this problem include: Fusobacterium nucleatum, Treponemadenticola, Tannerella forsythia (formerly Bacteroides forsythus),Prevotella intermedia, Porphyromonas gingivalis, Porphyromonasendodontalis, and Eubacterium species.

Dental plaque is a biofilm that forms on the surface of teeth. Oralcavity infections that are related to dental plaque include cariesdevelopment, gingivitis, and periodontitis. While hundreds of bacteriahave been detected in dental plaque, the most common bacteria thatcontribute to gingivitis and periodontitis are: Actinobacillusactinomycetemcomitans, Campylobacter rectus, Eikenella corrodens andseven anaerobic species, Porphyromonas gingivalis, Bacteroidesforsythus, Treponema denticola, Prevotella intermedia, Fusobacteriumnucleatum, Eubacterium, and spirochetes. P. gingivalis, a gram-negativeanaerobe, is believed to be largely responsible for adult periodontitis.Various herpes viruses have also been found to contribute to destructiveperiodontal disease. The bacteria that largely underlie caries formationare Streptococcus mutans, Lactobacillus acidophilus, Actinomycesviscosus, and Nocardia spp.

Oral thrush is the most common oral fungal infection. The causativeagents of oral thrush are Candida albicans and Candida dubliniensis. C.dubliniensis is typically found in immunocompromised patients, such asAIDS patients, organ transplant patients and patients undergoingchemotherapy.

Oral cavity infections such as gingivitis, periodontitis and caries arequite difficult to treat since, in many cases, the bacteria responsiblefor the infection are in biofilm form and located in difficult to reachsupra- and (even more difficult to reach) sub-gingival pockets.Periodontitis an inflammatory disease affecting the tissues thatsurround and support the teeth. The microorganism causing periodontitisadheres to and grows on the tooth's surfaces, and there is an overlyaggressive immune response against these microorganisms. Periodontitisinvolves progressive loss of the alveolar bone around the teeth, and, ifleft untreated, can lead to the loosening and subsequent loss of teeth.Standard treatment often involves root planning and scaling, which isuncomfortable for the patient and often requires an analgesic beadministered. In some cases, invasive surgery is necessary to treatperiodontitis. Current treatment often includes an antimicrobial mouthwash having chlorhexidine as the antimicrobial. Chlorhexidine can havethe undesirable side effect of staining teeth.

Thus, a method for alleviating infected oral cavity tissue is provided.The methods provided are contemplated as improving contact betweenchlorine dioxide and the reservoirs of biofilm located in supra- andsub-gingival pockets. Administration to the oral cavity can take theform of continuous irrigation, intermittant irrigation (e.g., rinsing orwashing), multiple substantially contiguous applications, combinationtherapy to improve tissue penetration, use of dental trays designed todeliver content to the gingival pockets, and direct tissue injection ofchlorine dioxide compositions such as gels or solutions. In someembodiments, a combination therapy is carried out, that includes a stepof improving oral mucosal tissue penetration of chlorine dioxide, forinstance by use of ultrasonic energy.

The methods described herein further generally pertain to theadministration of a composition comprising chlorine dioxide to a toothsurface in a substantially non-cytotoxic and/or non-irritating manner towhiten the tooth surface.

The normal shade of teeth is determined by the natural off-white tintsof the enamel and the dentin beneath. Extrinsic and intrinsic stainingalso contribute to tooth color. Extrinsic staining refers to surfacestains, such as those caused by tea, coffee, red wine, and other foodsrich in polyphones. Extrinsic stains can be removed through the use ofsurfactants and/or abrasives, which cause their physical removal fromthe tooth surface.

Intrinsic staining refers to stains that exist below enamel surface, orthat penetrate below enamel surface. Intrinsic staining can happen whenfood molecules seep into enamel flaws and cracks, or, in some cases,between enamel rods. Intrinsic discoloration can also occur following achange to the structural composition or thickness of the dental hardtissues. Removal of intrinsic staining is more difficult and timeconsuming than removal of extrinsic staining. Intrinsic stain removalcan be achieved by a variety of methods including use of peroxides orperoxide analogs, with or without chemical, light or heat activation, tobleach the stains. Among the side effects associated with the use ofperoxides are tooth sensitivity, soft tissue irritation (e.g., gingivalirritation) and tooth surface changes (e.g., changes to dentin and/orenamel).

Tooth whitening using substantially non-cytotoxic and/or substantiallynon-irritating chlorine dioxide-containing compositions has been shownto be efficacious in whitening and characterized by reduced soft tissueirritation and reduced changes to enamel and dentin of teeth. Seecommonly-assigned U.S. application Ser. Nos. 12/502,761, 12/502,781,12/502,895, 12/502,907, and 12/502,925, filed Jul. 14, 2009.

The methods described herein can provide statistically significantlyimproved tooth whitening. In an embodiment, the method for whitening atooth surface comprises at least two iterations of a step of contactingthe tooth surface with a composition comprising a chlorine dioxidesource, wherein the iterations are substantially contiguous. In anembodiment, each iteration uses a fresh specimen of the composition. By“fresh specimen” is meant a specimen of composition that has not beenpreviously exposed to a biological tissue. In one aspect, the contactingstep comprises contacting the tooth surface with a substantiallynon-cytotoxic and/or non-irritating composition. In another aspect, thecontacting step comprises irrigating the tooth surface using anirrigation device. The irrigation device can deliver a substantiallynon-cytotoxic and/or non-irritating fluid composition. Alternatively,the irrigation device can be modified so as to deliver a substantiallyoxy-chlorine anion free chlorine dioxide composition.

I. Chlorine Dioxide-Generating Components

The methods employ a composition that comprises a chlorine dioxidesource. A chlorine dioxide source refers to chlorine dioxide, chlorinedioxide-generating components, and combinations thereof. Chlorinedioxide-generating components refer to at least an oxy-chlorine anionsource and an activator of chlorine dioxide generation. In someembodiments, the activator is an acid source. In these embodiments, thecomponents optionally further includes a free halogen source. The freehalogen source may be a cationic halogen source, such as chlorine. Inother embodiments, the activator is an energy-activatable catalyst. Inyet other embodiments, the activator is a dry or anhydrous polarmaterial. In other embodiments, the activator is an aqueous fluid suchas water, saliva, mucus, and wound exudate, and/or water vapor.

Oxy-chlorine anion sources generally include chlorites and chlorates.The oxy-chlorine anion source may be an alkali metal chlorite salt, analkaline earth metal chlorite salt, an alkali metal chlorate salt, analkaline earth metal chlorate salt and combinations of such salts. Inexemplary embodiments, the oxy-chlorine anion source is a metalchlorite. In some embodiments, the metal chlorite is an alkali metalchlorite, such as sodium chlorite and potassium chlorite. Alkaline earthmetal chlorites can also be employed. Examples of alkaline earth metalchlorites include barium chlorite, calcium chlorite, and magnesiumchlorite. An exemplary metal chlorite is sodium chlorite.

For chlorine dioxide generation activated by an acid source, the acidsource may include inorganic acid salts, salts comprising the anions ofstrong acids and cations of weak bases, acids that can liberate protonsinto solution when contacted with water, organic acids, inorganic acids,and mixtures thereof. In some aspects, the acid source is a particulatesolid material which does not react substantially with the metalchlorite during dry storage, however, does react with the metal chloriteto form chlorine dioxide when in the presence of an aqueous medium. Theacid source may be water soluble, substantially insoluble in water, orintermediate between the two. Exemplary acid sources are those whichproduce a pH of below about 7, more preferably below about 5.

Exemplary substantially water-soluble, acid-source-forming componentsinclude, but are not limited to, water-soluble solid acids such as boricacid, citric acid, tartaric acid, water soluble organic acid anhydridessuch as maleic anhydride, and water soluble acid salts such as calciumchloride, magnesium chloride, magnesium nitrate, lithium chloride,magnesium sulfate, aluminum sulfate, sodium acid sulfate (NaHSO₄),sodium dihydrogen phosphate (NaH₂PO₄), potassium acid sulfate (KHSO₄),potassium dihydrogen phosphate (KH₂PO₄), and mixtures thereof. Exemplaryacid-source-forming component is sodium acid sulfate (sodium bisulfate).Additional water-soluble, acid-source-forming components will be knownto those skilled in the art.

Chlorine dioxide-generating components optionally comprise a source offree halogen. In one embodiment, the free halogen source is a freechlorine source, and the free halogen is free chlorine. Suitableexamples of free halogen source used in the anhydrous compositionsinclude dichloroisocyanuric acid and salts thereof such as NaDCCA,trichlorocyanuric acid, salts of hypochlorous acid such as sodium,potassium and calcium hypochlorite, bromochlorodimethylhydantoin,dibromodimethylhydantoin and the like. An exemplary source of freehalogen is NaDCCA.

For chlorine dioxide generation activated by an energy-activatablecatalyst, the energy-activatable catalyst can be selected from the groupconsisting of a metal oxide, a metal sulfide, and a metal phosphide.Exemplary energy-activatable catalysts include metal oxides selectedfrom the group consisting of titanium dioxide (TiO₂); zinc oxide (ZnO);tungsten trioxide (WO₃); ruthenium dioxide (RuO₂); iridium dioxide(IrO₂); tin dioxide (SnO₂); strontium titanate (SrTiO₃); barium titanate(BaTiO₃); tantalum oxide (Ta₂O₅); calcium titanate (CaTiO₃); iron (III)oxide (Fe₂O₃); molybdenum trioxide (MoO₃); niobium pentoxide (NbO₅);indium trioxide (In₂O₃); cadmium oxide (CdO); hafnium oxide (HfO₂);zirconium oxide (ZrO₂); manganese dioxide (MnO₂); copper oxide (Cu₂O);vanadium pentoxide (V₂O₅); chromium trioxide (CrO₃); yttrium trioxide(YO₃); silver oxide (Ag₂O), Ti_(x)Zr_(1-x)O₂, wherein x is between 0 and1, and combinations thereof. The energy-activatable catalyst can beselected from the group consisting of titanium oxide, zinc oxide,calcium titanate, zirconium oxide and combinations thereof.

Chlorine dioxide-generating components optionally may be present in amatrix. Such matrices may be organic matrices, such as those describedin commonly-assigned U.S. Pat. Publication No. 2006/0024369. In thesematrices, chlorine dioxide is generated when the composite is exposed towater vapor or electromagnetic energy. The matrix may be a hydrous gelor an anhydrous gel. Hydrophobic matrices may also be employed.Hydrophobic matrix materials include water-impervious solid componentssuch as hydrophobic waxes, water-impervious fluids such as hydrophobicoils, and mixtures of hydrophobic solids and hydrophobic fluids. Inembodiments using a hydrophobic matrix, activation of chlorine dioxidemay be a dry or anhydrous polar material, as described in co-pendingU.S. Application No. 61/153,847.

II. Delivery Methods

1. Non-Cytotoxic/Non-Irritating Composition

In an embodiment, the method comprises contacting a wound, toothsurface, or an oral cavity with a substantially non-cytotoxic and/orsubstantially non-irritating composition comprising a chlorine dioxidesource.

For compositions comprising an oxidizing or bleaching agent consistingof chlorine dioxide, cytotoxicity results predominantly from thepresence of oxy-chlorine anions, absent other constituents thatcontribute to cytotoxicity. Accordingly, a composition comprisingchlorine dioxide that comprises zero milligram (mg) oxy-chlorine anionper gram composition to no more than about 0.25 mg oxy-chlorine anionper gram composition, from zero to about 0.24, 0.23, 0.22, 0.21, or 0.20mg oxy-chlorine anion per gram composition, from zero to 0.19, 0.18,0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, or 0.10 mg oxy-chlorine anionper gram composition or from zero to 0.09, 0.08, 0.07, 0.06, 0.05 or0.04 mg oxy-chlorine anion per gram composition, absent otherconstituents that contribute to cytotoxicity, is substantiallynon-cytotoxic.

Oxy-chlorine anions can be measured in chlorine dioxide solutions orcompositions using any method known to those skilled in the art,including ion chromatography following the general procedures of EPAtest method 300 (Pfaff, 1993, “Method 300.0 Determination of InorganicAnions by Ion Chromatography,” Rev. 2.1, US Environmental ProtectionAgency) or a titration method based on an amperometric method(Amperometric Method II in Eaton et al, ed., “Standard Methods for theExamination of Water and Wastewater” 19^(th) edition, American PublicHealth Association, Washington D.C., 1995). Alternatively, oxy-chlorineanions may be measured by a titration technique equivalent to theamperometric method, but which uses the oxidation of iodide to iodineand subsequent titration with sodium thiosulfate to a starch endpoint inplace of the amperometric titration; this method is referred to hereinas “pH 7 buffered titration.” A chlorite analytical standard can beprepared from technical grade solid sodium chlorite, which is generallyassumed to comprise about 80% by weight of pure sodium chlorite.

Soft tissue irritation can result from highly reactive oxygen speciesand/or extremes of pH, both acidic and basic. To minimize soft tissueirritation by the chlorine dioxide containing composition, thesubstantially non-cytotoxic composition has a pH of at least 3.5.Preferably, the composition has a pH of at least 5, and more preferablystill, greater than about 6. In certain embodiments, the pH ranges fromabout 4.5 to about 11, more preferably from about 5 to about 9, and morepreferably still, greater than about 6 and less than about 8. In oneembodiment, the pH is about 6.5 to about 7.5. The concentration ofoxy-chlorine anions is not believed to be a primary contributor to softtissue irritation.

Methods of preparing non-cytotoxic and/or non-irritating compositionscomprising chlorine dioxide are described in commonly-assigned U.S.application Ser. Nos. 12/502,761 and 12/502,781, filed Jul. 14, 2009,entitled “Tooth Whitening Compositions and Methods,” and U.S.application Ser. Nos. 12/502,326 and 12/502,356, filed Jul. 14, 2009,entitled “Non-Cytotoxic Chlorine Dioxide Fluids,” each of which isincorporated herein by reference in its entirety.

In an embodiment, a substantially non-cytotoxic composition comprisingchlorine dioxide can be prepared using a substantially pure chlorinedioxide solution having a neutral pH. In some embodiments, thesubstantially pure chlorine dioxide solution has a pH from about 5 toabout 9, and more preferably, from about 6.5 to about 7.5.

Substantially pure chlorine dioxide may be prepared by preparing achlorine dioxide solution using any known method, then bubbling a gas(e.g., air) through that solution (sparging) and into a second containerof deionized water, to prepare the product solution of substantiallypure chlorine dioxide. Only ClO₂ and possibly some water vapor aretransferred from the source solution to the product solution. All thesalt ingredients and acid remain behind in the source solution. Thus,there are no oxy-chlorine anions in the substantially pure productsolution. One method of preparing chlorine dioxide comprises combiningan aqueous solution of sodium chlorite with a mineral acid to reduce thesolution pH to below about 3.5 and allowing the solution to react for asufficient time, e.g., about 30 minutes, to generate chlorine dioxide.The resulting solution is then sparged, as described above, to preparethe product solution of substantially pure chlorine dioxide.

While the substantially pure chlorine dioxide may undergo a degree ofdecomposition, the rate is relatively slow. By keeping the solutioncapped and protected from ultraviolet exposure, the decomposition ratecan be slowed to a rate of about 5% to about 25% reduction in chlorinedioxide in 7 days. Substantially pure chlorine dioxide may also beprepared using a pervaporation technique, such as that disclosed in U.S.Pat. No. 4,683,039. In addition, a metal chlorite and an acid source canbe reacted in solution to yield high conversion to chlorine dioxide andproduce a greater than 2000 ppm chlorine dioxide solution. Theconcentrated solution can then be buffered to a neutral pH. Similarly, achlorine dioxide solution can be prepared using the compositiondescribed in U.S. Pat. No. 5,399,288, which yields a high concentrationchlorine dioxide solution at acidic pH. The concentrated solution canthen be buffered to achieve a substantially neutral pH to prepare asubstantially pure chlorine dioxide solution.

Another source of a substantially pure chlorine dioxide solution ischlorine dioxide is prepared using an ASEPTROL (BASF Corp., FlorhamPark, N.J.) material, which are described in commonly-assigned U.S. Pat.Nos. 6,432,322 and 6,699,404. These patents disclose substantiallyanhydrous solid bodies comprising particulate components for preparinghighly-converted solutions of chlorine dioxide when added to water. Theparticulate components in the solid bodies comprise a metal chloritesuch as sodium chlorite, an acid source such as sodium bisulfate andoptionally a source of free halogen such as the sodium salt ofdichloroisocyanuric acid or a hydrate thereof (collectively referred toherein as “NaDCCA”). Chlorine dioxide is generated when an ASEPTROLmaterial is contacted with water or an aqueous medium. ASEPTROL materialcan be made to have an extremely high conversion rate in an aqueoussolution, as described in U.S. Pat. Nos. 6,432,322 and 6,699,404,resulting in high concentrations of chlorine dioxide and lowconcentrations of oxy-chlorine anion. Thus, ASEPTROL materials provide away to efficiently generate chlorine dioxide at substantially neutralpH, thus avoiding problems existing with earlier, acidic chlorinedioxide-based products.

In some embodiments, the composition further comprises a thickenercomponent which renders the composition a thickened aqueous fluid. Toprepare a thickened aqueous composition comprising chlorine dioxide thatis substantially non-cytotoxic and, in some embodiments, non-irritating,the substantially pure chlorine dioxide solution can be combined with athickener component and an aqueous medium.

The aqueous thickened fluid composition used in practicing the methodmay comprise any thickener component in an aqueous medium, wherein thethickened fluid composition is non-cytotoxic and/or non-irritating tosoft tissues. In exemplary embodiments, the thickener is not adverselyaffected by the chlorine dioxide on the time scale of compositionpreparation and use in treatment. Many thickener agents are known in theart, including, but not limited to carbomers (e.g., CARBOPOL thickeners,Lubrizol Corp., Wickliffe, Ohio), sodium carboxymethylcellulose (NaCMC),ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose(HPMC), natural smectite clays (e.g., VEEGEM, R.T. Vanderbilt Co.,Norwalk, Conn.), synthetic clays (e.g., LAPONITE (Southern ClayProducts, Gonzales, Tex.), methylcellulose, superabsorbent polymers suchas polyacrylates (e.g., LUQUASORB 1010, BASF, Florham Park, N.J.),poloxamers (PLURONIC, BASF, Florham Park, N.J.), polyvinyl alcohol,sodium alginate, tragacanth, and xanthan gum. Such thickening agents maybe categorized into four groups: natural hydrocolloids (also referred toas “gum”), semisynthetic hydrocolloids, synthetic hydrocolloids, andclay. Some examples of natural hydrocolloids include acacia, tragacanth,alginic acid, carrageenan, locust bean gum, guar gum, and gelatin.Non-limiting examples of semisynthetic hydrocolloids includemethylcellulose and sodium carboxymethylcellulose. Some examples ofsynthetic hydrocolloids (also referred to as “polymers” includingpolymers, cross-linked polymers, and copolymers) include polyacrylates,superabsorbent polymers, high molecular weight polyethylene glycols andpolypropylene glycols, polyethylene oxides and CARBOPOL. Non-limitingexamples of clay (including swelling clay) include LAPONITE,attapulgite, bentonite, and VEEGUM. In some embodiments, the thickenercomponent can be a semisynthetic hydrocolloid. An exemplary thickenercomponent is hydroxypropyl methylcellulose or a carboxymethylcellulose(CMC).

In preparing a non-cytotoxic composition, one or more components of acomposition may be combined prior to the time of preparation of acomposition. Alternatively, all components of a composition may beprepared at the time of use. For either non-cytotoxic solutions ornon-cytotoxic thickened compositions, optional other components suitablefor the intended use of the non-cytotoxic chlorine dioxide solution, asdescribed elsewhere herein, may be included. Chlorine dioxide insolution will decompose over time. To avoid problems arising from suchdecomposition, including loss of efficacy and generation of chloriteanions, the substantially pure chlorine dioxide solution is preferablyprepared immediately before its dilution or its combination with athickener component and an aqueous medium.

In addition, in some embodiments, a thickened composition comprisingchlorine dioxide can be prepared immediately before its use.“Immediately before” as used herein, refers to a period no greater thanthat which would result in diminished efficacy or evidence ofcytotoxicity. Generally, “immediately before” is less than about 14days, and preferably no greater than about 24 hours and more preferablyno greater than about 2 hours. In exemplary embodiments, thesubstantially pure chlorine dioxide solution is prepared within about 8hours of the preparation of the composition. Precautions are also takento avoid exposing the chlorine dioxide solution or the preparedcomposition to strong ultraviolet light or elevated temperature (e.g.,temperature greater than ambient temperature, about 25° C.).

A substantially non-cytotoxic thickened composition comprising chlorinedioxide may also be prepared using a particulate precursor of ClO₂ andan aqueous thickened fluid composition. Chlorine dioxide-formingcomponents include metal chlorites, metal chlorates, an acid source, andan optional halogen source. The particulate precursor may comprise oneof these or any combination of these. An exemplary particulate precursoris an ASEPTROL product. An exemplary ASEPTROL product is ASEPTROLS-Tab2. ASEPTROL S-Tab2 has the following chemical composition by weight(%): NaClO₂ (7%); NaHSO₄ (12%); NaDCC (1%); NaCl (40%); MgCl₂ (40%).Example 4 of U.S. Pat. No. 6,432,322 describes an exemplary manufactureprocess of S-Tab2. Granules can be produced, either by comminutingpressed S-Tab2 tablets, or by dry roller compaction of the non-pressedpowder of the S-Tab2 components, followed by breakup of the resultantcompacted ribbon or briquettes, and then screening to obtain the desiredsize granule. Upon exposure to water or an aqueous thickened fluid,chlorine dioxide is generated from the ASEPTROL granules. In oneembodiment, a substantially non-cytotoxic composition comprisingchlorine dioxide is prepared by combining −40 mesh granules with anaqueous thickened fluid. In one embodiment, the thickener component ofthe thickened fluid is carboxymethylcellulose or HPMC. The skilledartisan will recognize that chlorine dioxide production in the thickenedfluid composition prepared using a particulate precursor of ClO₂, whilerapid, is not instantaneous. Thus, sufficient time for the generation ofchlorine dioxide, and corresponding consumption of chlorite anion, isnecessary to obtain a substantially non-cytotoxic thickened fluidcomposition. The skilled artisan can readily determine what length oftime is sufficient, in view of the teachings in this disclosure and theknowledge of the art.

In some embodiments, the aqueous thickened fluid is preparedsufficiently in advance of combining with the ASEPTROL granules toenable the complete hydration of the thickener component. In oneembodiment, the thickened fluid composition is formed by adding highviscosity NaCMC powder to distilled water. The NaCMC is allowed tohydrate for at least 8 hours, and then the mixture is stirred tohomogenize it. A substantially non-cytotoxic composition is thenprepared by mixing the sized ASEPTROL granules with the NaCMC thickenedfluid. Contact with the aqueous medium in the hydrated NaCMC mixtureactivates the ASEPTROL granules and chlorine dioxide is generated.

In another embodiment, the substantially non-cytotoxic thickened fluidcomposition may also be formed at the site of intended use. Forinstance, a body fluid such as mucus of mucosal tissue, saliva, woundexudate or humid vapor such as exhaled air, may serve as the aqueousmedium to activate particulate precursors of chlorine dioxide, such asASEPTROL granules. In one embodiment, the mixture may be particulates inthe form of a powder and mixed in a layer of thickener component therebyforming a thickened matrix. The matrix may be applied directly to amucosal tissue or wound, wherein exposure to moisture present in thetissue activates production of chlorine dioxide to form a substantiallynon-cytotoxic composition. Alternatively, the matrix may be moistenedimmediately prior to use and then applied to any tissue.

In an embodiment, the substantially non-cytotoxic and/or non-irritatingcomposition consists essentially of chlorine dioxide as the activepharmaceutical ingredient (API). In other embodiments, the compositioncomprises chlorine dioxide in combination with at least one other API,such as an antimicrobial, topical anesthetic, topical analgesic, steroidor combination thereof. The composition optionally comprises one or moreother components. Such components include, but are not limited to,coloring agents and fragrances. Other optional components include:enzymes, malodor controlling agents, and the like. Exemplaryantimicrobials include, but are not limited to, gatifloxacin,clindamycin, gentiamicin, ceftazidime, an aminoglycoside such astobramyin and streptomycin, amphotericin B, itraconazole, ketoconazole,miconazole, nystatin, neomycin, ribaximin, clindamycin, metronidazole,polymixin B, proguanil, econazole, and fluconazole.

In some embodiments, all optional components are relatively resistant tooxidation by chlorine dioxide, since oxidation of composition componentsby chlorine dioxide will reduce the available chlorine dioxide foroxidation for its intended function. “Relatively resistant” means thatin the time scale of preparing and using the chlorine dioxide-containingcomposition in an application, the function of the optional component isnot unacceptably diminished, and the composition retains an acceptablelevel of efficacy/potency with respect to the chlorine dioxide andremains substantially non-cytotoxic. In some embodiments, thecomposition can remain substantially non-irritating. Guidance regardingidentifying resistant components is provided in commonly-assigned U.S.patent application entitled “Additives for Chlorine Dioxide-ContainingCompositions,” serial number 61/299,999, filed Jan. 31, 2010.

The substantially non-cytotoxic and/or substantially non-irritatingcomposition can be delivered to tissue using any method known in theart. Formulations can include ointment, gel, cream, lotion, plasters,transdermal patches, inserts, rinses, and the like. For oralapplications, the composition can also be delivered to a tooth surfaceor oral tissue by dental tray, dental film, or dental strip. A dentalstrip refers to a substantially planar object made of a plastic backbonethat is sufficiently flexible to affix to teeth. A dental film refers toa substantially planar object made of a pliable, conformable materialthat can be substantially fitted to the surface of teeth. Optionally,the dental strip is dissolvable in an aqueous medium, such as saliva.

2. Devices and Compositions for Non-Cytotoxic Administration

In home embodiments, the method can be practiced with a device orcomposition that delivers a substantially oxy-chlorine anion freechlorine dioxide composition to the wound, tooth surface, or to theinfected oral cavity tissue. Such devices, compositions, systems andmethods for administration of a composition comprising chlorine dioxideand oxy-chlorine anions in a way that the chlorine dioxide reaches thetarget tissue in an efficacious amount, but the oxy-chlorine anions aresubstantially inhibited from irritating target tissue or peripheraltissue not targeted for treatment, are described in commonly-assignedU.S. application Ser. Nos. 12/502,845, 12/502,858 and 12/502,877, filedJul. 14, 2009, entitled “Methods, Systems and Devices for Administrationof Chlorine Dioxide.” Generally, the method comprises providing achlorine dioxide source that includes either chlorine dioxide itself orchlorine dioxide-generating components, and further includes theoxy-chlorine anions that cause cytotoxicity to tissues; and furtherproviding an oxy-chlorine anion barrier that substantially prohibitspassage therethrough of the oxy-chlorine anions and permits passagetherethrough of chlorine dioxide. In some embodiments, the oxy-chlorineanion barrier can also substantially inhibit the passage therethrough ofprotons. The chlorine dioxide source is applied to the tissue with theoxy-chlorine anion barrier interposed between the chlorine dioxidesource and the tissue, thus preventing or substantially minimizing theoxy-chlorine anion from reaching the tissue, thereby enabling deliveryof a substantially oxy-chlorine anion free chlorine dioxide compositionto the tissue.

The chlorine dioxide that comes into contact with the tissue issubstantially oxy-chlorine anion free. In one embodiment, thesubstantially oxy-chlorine anion free chlorine dioxide that contacts thetissue comprises zero milligram (mg) oxy-chlorine anion per gram to nomore than about 0.25 mg oxy-chlorine anion per gram, or from zero to0.24, 0.23, 0.22, 0.21, or 0.20 mg oxy-chlorine anion per gramcomposition, or from zero to 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13,0.12, 0.11, or 0.10 mg oxy-chlorine anion per gram composition, or fromzero to 0.09, 0.08, 0.07, 0.06, 0.05 or 0.04 mg oxy-chlorine anion pergram composition, absent other constituents that contribute tocytotoxicity, and is therefore substantially non-cytotoxic. In someembodiments, the substantially oxy-chlorine anion free chlorine dioxidecomprises less than about 400 milligrams per square meter of contactarea, less than about 375 mg/m², less than about 350 mg/m², than about325 mg/m², or than about 300 mg/m² oxy-chlorine anions. In someembodiments, the substantially oxy-chlorine anion free chlorine dioxidecomprises from zero to less than about 200 mg/m²oxy-chlorine anions. Inother embodiments, the substantially oxy-chlorine anion free chlorinedioxide comprises from zero to less than about 100 mg/m²oxy-chlorineanions.

The chlorine dioxide source can comprise any chlorine dioxide-containingcomposition or ingredients capable of forming chlorine dioxide in situ.In exemplary embodiments, the ingredients present in the chlorinedioxide source are compatible with the oxy-chlorine anion barrier duringthe practice of the method, as well as any pre-use period during whichthe ingredients are in contact with the barrier. By “compatible” ismeant the ingredients do not adversely affect to an unacceptable degreethe concentration of chlorine dioxide in the chlorine dioxide source,the inhibition of passage of oxy-chlorine anions, or the permittedpassage of chlorine dioxide by the barrier.

The barrier may be in the form of a layer between the chlorine dioxidesource and the target tissue. In one aspect, the oxy-chlorine barrier,without the chlorine dioxide source, is applied to the tissue first. Thechlorine dioxide source is then applied to the barrier layer. In otherembodiments, the chlorine dioxide source can be applied to the barrierfirst, and the combination can be then applied to the tissue, whereinthe barrier layer contacts the tissue. In embodiments where the chlorinedioxide source comprises chlorine dioxide-generating components, thegeneration of chlorine dioxide may be activated before, during, and/orafter application of the barrier (with or without the chlorine dioxidesource) to the target tissue.

In another embodiment, the target tissue may be contacted with achlorine dioxide source containing a substantially non-cytotoxic andsubstantially non-irritating amount of oxy-chlorine anions while asecond chlorine dioxide source may be located on the side of a barrieropposite the target tissue such that additional chlorine dioxide fromthe second source may pass through the barrier to contact the targettissue but passage through the barrier of oxy-chlorine anions in thesecond source is inhibited.

In another embodiment, the chlorine dioxide source may be dispersed in amatrix comprising one or more barrier substances, such that theoxy-chlorine anions are sequestered away from the tissue, while thechlorine dioxide passes through the barrier substance, if necessary, andthe matrix to contact the target tissue. In this embodiment, the matrixis applied to the tissue directly or to an optional interveningtissue-contacting layer. In one aspect, the matrix itself is the barriersubstance. Exemplary matrix materials that may also function as thebarrier include waxes such as paraffin wax, polyethylene, petrolatum,polysiloxanes, polyvinyl alcohol, ethylene-vinyl acetate (EVA),polyurethanes, mixtures thereof, and the like. In another aspect, thechlorine dioxide source can be coated or encapsulated by the barriersubstance. Exemplary barrier substances include polyurethane,polypropylene, polytetrafluoroethylene, polyvinylidene difluoride,polyvinylidene dichloride, combination of polydimethylsiloxane andpolytetrafluoroethylene, polystyrene, cellulose acetate, polysiloxane,polyethylene oxide, polyacrylates, mineral oil, paraffin wax,polyisobutylene, polybutene and combinations thereof. Exemplary barriersubstances also comprise compounds that bind to oxy-chlorine anions withhigh affinity and that impede or stop anion migration or diffusion suchthat a substantially oxy-chlorine anion free chlorine dioxidecomposition can be delivered to a tissue. The compound may form aninsoluble precipitate with the oxy-chlorine anion, thereby impeding orstopping diffusion. Alternatively, the compound is immobilized on asubstance or material, thereby impeding diffusion or migration. Thecompound may be cationic, such as ammonium, pyridinium, imidazolium,phosphonium, and sulfonium, and other positively charged compounds thatmay be part of the matrix. Optionally, the compound can be immobilizedon an oxy-chlorine anion barrier material, to the matrix or on theoptional backing layer.

Various materials and membranes can be used as an oxy-chlorine anionbarrier. The barrier can be in any form, and is typically either a fluidor a solid.

In some embodiments, the oxy-chlorine anion barrier is a fluid, such apetrolatum. In this embodiment, the fluid can be applied to the tissuefirst, or to an intervening tissue-contacting layer, to form the barrieras a layer and then chlorine dioxide source subsequently applied to thefluid barrier layer. The chlorine dioxide source can be applied as aparticulate or can be encompassed in a material to form a film.

In some embodiments, the oxy-chlorine anion barrier is a nonporousmembrane. The membrane can be any thickness and can be a single layer orplural layers, provided the membrane remains permeable to chlorinedioxide and substantially non-permeable to oxy-chlorine anions. Anexemplary nonporous material is a polyurethane membrane. In someembodiments, the polyurethane membrane is from about 30 to about 100microns, such as from about 38 to about 76 microns thick. Exemplarypolyurethane membranes commercially available include CoTran™ 9701 (3M™Drug Delivery Systems, St. Paul, Minn.) and ELASTOLLAN (BASF Corp.,Wyandotte, Mich.). ELASTOLLAN products are polyether-based thermoplasticpolyurethane. A specific example of ELASTOLLAN is ELASTOLLAN 1185A10.

In some embodiments, the oxy-chlorine anion barrier is a microporousmembrane permeable to chlorine dioxide and substantially non-permeableto oxy-chlorine anions. The microporous membrane can be any thicknessand can be a single layer or plural layers, provided the membraneremains permeable to chlorine dioxide and substantially non-permeable tooxy-chlorine anions. In one example, the microporous membrane cancomprise thermo-mechanically expanded polytetrafluoroethylene (e.g.,Goretex®) or polyvinylidenedifluoride (PVDF). See, for instance, U.S.Pat. No. 4,683,039. The procedure for formation of an expandedpolytetrafluoroethylene is described in U.S. Pat. No. 3,953,566. Anexemplary polytetrafluoroethylene (PTFE) membrane, interpenetratingpolymer network (IPN) of polydimethylsiloxane and PTFE, is described inU.S. Pat. Nos. 4,832,009, 4,945,125, and 5,980,923. Acommercially-available product of this type, Silon-IPN (Bio Med SciencesInc., Allentown, Pa.), is a single layer and is available in thicknessesbetween 10 to 750 microns. In one embodiment, the microporous membraneis an IPN of silicone and PTFE having a thickness of about 16 microns.In another example, the membrane is microporous polypropylene film. Anexemplary microporous polypropylene film is the materialcommercially-available from CHEMPLEX Industries (Palm City, Fla.), whichis a single layer membrane about 25 microns thick, having a porosity of55% and a pore size of about 0.21 microns×0.05 microns. The microporousmembrane material can be provided as a composite with supportingmaterials to provide the structural strength required for use. In someembodiments, the membrane is hydrophobic, wherein the hydrophobic natureof the membrane prevents both an aqueous reaction medium and an aqueousrecipient medium from passing through the membrane, while allowingmolecular diffusion of chlorine dioxide. Features to consider for thematerials used for such a barrier include: hydrophobicity of themicroporous material, pore size, thickness, and chemical stabilitytowards the attack of chlorine dioxide, chlorine, chlorite, chlorate,chloride, acid, and base.

Various other materials and membranes can be used to form the barrier.For example, the barrier can comprise a microperforated polyolefinmembrane; a polystyrene film that is substantially permeable to chlorinedioxide and substantially impermeable to ionic components of thecomposition; a pervaporation membrane formed from a polymeric materialhaving a relatively open polymeric structure; a cellulose acetate filmcomposite; a polysiloxane or polyurethane material; or a wax. Of course,for contact with mucosal or dermal tissues, the microporous barriershould be substantially non-irritating and substantially non-cytotoxic,particularly in the time scale of typical use of the device andcomposition.

The pore sizes in the barrier may vary widely, depending on the desiredflow rate of the chlorine dioxide through the barrier. The pores shouldnot be so small as to prevent chlorine dioxide gas flow therethrough butalso should not be so large that liquid flow is permitted. In oneembodiment, the pore size is about 0.21 microns×0.05 microns. Thequantity and size of the pores of the barrier can vary widely, dependingupon the temperature of the application, the hydrophobicity of thebarrier material, the thickness of the barrier material, and alsodepending upon the desired flow rate of chlorine dioxide through thebarrier. Fewer and smaller pores are needed for a given chlorine dioxideflow rate at higher temperature relative to lower temperature, as thevapor pressure of chlorine dioxide from the chlorine dioxide source ishigher at the higher temperature. More and larger pores can be used witha highly hydrophobic barrier material, such as PTFE, compared to a lesshydrophobic material, such as polyurethane, since the tendency for anaqueous chlorine dioxide source to flow through pores of a highlyhydrophobic barrier is lower than it is through the pores of a lesshydrophobic barrier. Considerations of barrier strength also dictate theporosity chosen. Generally, the barrier porosity varies from about 1 toabout 98%, from about 25 to about 98%, or from about 50% to about 98%.

Also provided are systems, compositions, and devices useful forpracticing the method. In one aspect, a system is provided fordelivering a substantially oxy-chlorine anion free chlorine dioxide to atissue. A typical system comprises a chlorine dioxide source thatincludes chlorine dioxide or chlorine dioxide-generating components, andoxy-chlorine anions as a first system component; and an oxy-chlorineanion barrier as a second system component, the barrier to be interposedbetween the chlorine dioxide source and the tissue, wherein the barriersubstantially prohibits passage of the oxy-chlorine anions and permitspassage of the substantially oxy-chlorine anion free chlorine dioxidecomposition, thereby enabling delivery of the substantially oxy-chlorineanion free chlorine dioxide to the tissue.

Compositions and devices are also provided to implement the methods andsystems described above. Thus, one aspect features a composition fordelivering a substantially oxy-chlorine anion free chlorine dioxidecomposition to a tissue. The composition comprises a matrix thatincludes a chlorine dioxide source comprising chlorine dioxide orchlorine dioxide-generating components, as well as oxy-chlorine anions,and at least one barrier substance that substantially prohibits passageof the oxy-chlorine anions but permits passage of the chlorine dioxide,thereby enabling delivery of the substantially oxy-chlorine anion freechlorine dioxide to the tissue. In one embodiment, the matrix can be aaqueous matrix, or a hydrophobic or anhydrous matrix such as petrolatum.In some embodiments, the matrix itself is the barrier substance. Forinstance, the matrix can be nonpolar or weakly polar for inhibitingdiffusion of oxy-chlorine anions while permitting diffusion of chlorinedioxide.

The bulk of the matrix can be the barrier substance, or the matrix cancomprise a sufficient amount of the barrier substance to carry out theselective delivery of the chlorine dioxide to the tissue. For instance,the matrix can comprise a polymeric material in which reactants orprecursors for the formation of chlorine dioxide are embedded ordispersed, wherein the polymeric material is permeable to chlorinedioxide but substantially impermeable to oxy-chlorine anions. See, e.g.,U.S. Pat. No. 7,273,567, which describes a composition comprisingreactants or precursors and an energy-activatable catalyst embedded inpolyethylene, which are activated to produce chlorine dioxide byexposure to light waves, and more particularly, by exposure toultraviolet radiation.

In some embodiments, the matrix can be an adhesive matrix, such as anadhesive polymer matrix. Polymers useful in such adhesive matrices aresubstantially permeable to chlorine dioxide and are preferablyrelatively resistant to oxidation by chlorine dioxide so as to limitpossible degradation of the polymer and possible consequential change inadhesion. Adhesive polymers are known in the art. See, e.g., U.S. Pat.No. 7,384,650.

The composition can be applied to the tissue, e.g., by spreading it onor otherwise applying it to the tissue, or by incorporating it into adelivery device, such as described below.

Various delivery devices are envisioned for delivering a compositioncomprising chlorine dioxide and oxy-chlorine anions to target tissuesuch that an efficacious amount of chlorine dioxide contacts the targettissue, while the oxy-chlorine anions are substantially inhibited orprevented from contacting the tissue. The substantial inhibitionreduces, minimizes, or precludes damage or irritation to, the targettissue and any surrounding or peripheral tissues.

The devices are typically directionally oriented to comprise a layerdistal to the tissue to be contacted and a layer proximal to the tissueto be contacted. The distal layer is also referred to herein as abacking layer. The devices may further comprise a release liner affixedto the tissue-contacting layer, to be removed prior to applying thedevice to the tissue. In one embodiment, the device comprises a layercomprising the chlorine dioxide source and a barrier layer. In anotherembodiment, the device comprises (1) a backing layer, (2) a layercomprising the chlorine dioxide source, and (3) a barrier layer. Thebarrier layer can be adapted to contact the non-oral tissue, or anothertissue-contacting layer may be present between the barrier layer and thetissue. The barrier layer or the additional tissue-contacting layer canbe adhesive. The optional additional tissue-contacting layer is alsosubstantially permeable to chlorine dioxide. In some embodiments, thebarrier layer can be made from a thermo-mechanically expandedpolytetrafluoroethylene film. In some embodiments, the chlorine dioxidesource is a particulate precursor of chlorine dioxide, such as granulesof ASEPTROL.

Generally, the backing layer can be made of any suitable material thatis substantially impermeable to chlorine dioxide and other components ofthe chlorine dioxide source. The backing layer can serve as a protectivecover for the matrix layer and can also provide a support function.Exemplary materials for the backing layer include films of high andlow-density polyethylene, polyvinylidene dichloride (PVDC),polyvinylidene difluoride (PVDF), polypropylene, polyurethane, metalfoils, and the like.

The optional tissue-contacting layer can be any material that issubstantially permeable to chlorine dioxide. The optionaltissue-contacting layer can be an absorbent material. Non-limitingexamples for this layer include cotton or other natural fiber orsynthetic fiber fabrics or meshes, foams and mats.

In another embodiment, the device comprises a backing layer and a matrixas described above, in which is dispersed the chlorine dioxide sourceand which comprises at least one barrier substance. The matrix can beadapted for contacting the tissue, or an additional tissue-contactinglayer may be present. Either the matrix or the additionaltissue-contacting layer can be adhesive. Typically, the matrix isprepared and then coated onto the backing layer.

Any method in the art for preparing chlorine dioxide may be used as thechlorine dioxide source to make chlorine dioxide in the devices andcompositions that deliver a substantially oxy-chlorine anion freechlorine dioxide composition. For instance, there are a number ofmethods of preparing chlorine dioxide by reacting chlorite ions in waterto produce chlorine dioxide gas dissolved in water. The traditionalmethod for preparing chlorine dioxide involves reacting sodium chloritewith gaseous chlorine (Cl₂(g)), hypochlorous acid (HOCl), orhydrochloric acid (HCl). However, because the kinetics of chlorinedioxide formation are high order in chlorite anion concentration,chlorine dioxide generation is generally done at high concentration(>1000 ppm), the resulting chlorine dioxide containing solutiontypically must be diluted for the use concentration of a givenapplication. Chlorine dioxide may also be prepared from chlorate anionby either acidification or a combination of acidification and reduction.Chlorine dioxide can also be produced by reacting chlorite ions withorganic acid anhydrides.

Chlorine dioxide-generating compositions, which are comprised ofmaterials that will generate chlorine dioxide gas upon contact withwater vapor, are known in the art. See, e.g., commonly-assigned U.S.Pat. Nos. 6,077,495; 6,294,108; and 7,220,367. U.S. Pat. No. 6,046,243discloses composites of chlorite salt dissolved in a hydrophilicmaterial and an acid releasing agent in a hydrophobic material. Thecomposite generates chlorine dioxide upon exposure to moisture.Commonly-assigned U.S. Pat. Publication No. 2006/0024369 discloses achlorine dioxide-generating composite comprising a chlorinedioxide-generating material integrated into an organic matrix. Chlorinedioxide is generated when the composite is exposed to water vapor orelectromagnetic energy. Chlorine dioxide generation from a dry oranhydrous chlorine dioxide-generating composition by activation with adry polar material is disclosed in commonly-assigned co-pending U.S.Provisional Application No. 61/153,847. U.S. Pat. No. 7,273,567describes a method of preparing chlorine dioxide from a compositioncomprising a source of chlorite anions and an energy-activatablecatalyst. Exposure of the composition to the appropriate electromagneticenergy activates the catalyst which in turn catalyzes production ofchlorine dioxide gas.

Chlorine dioxide solutions can also be produced from solid mixtures,including powders, granules, and solid compacts such as tablets andbriquettes, which are comprised of components that will generatechlorine dioxide gas when contacted with liquid water. See, forinstance, commonly-assigned U.S. Pat. Nos. 6,432,322; 6,699,404; and7,182,883; and U.S. Pat. Publication Nos. 2006/0169949 and 2007/0172412.In some embodiments, chlorine dioxide is generated from a compositioncomprising a particulate precursor of chlorine dioxide. Thus, thechlorine dioxide source comprises or consists essentially of aparticulate precursor of chlorine dioxide. The particulate precursoremployed can be an ASEPTROL product, such as ASEPTROL S-Tab2 andASEPTROL S-Tab10. ASEPTROL S-Tab2 has the following chemical compositionby weight (%): NaClO₂ (7%); NaHSO₄ (12%); sodium dichloroisocyanuratedihydrate (NaDCC) (1%); NaCl (40%); MgCl₂ (40%). Example 4 of U.S. Pat.No. 6,432,322 describes an exemplary manufacture process of S-Tab2tablets. ASEPTROL S-Tab10 has the following chemical composition byweight (%): NaClO₂ (26%); NaHSO₄ (26%); NaDCC (7%); NaCl (20%); MgCl₂(21%). Example 5 of U.S. Pat. No. 6,432,322 describes an exemplarymanufacture process of S-Tab10 tablets.

As described elsewhere herein, activation of chlorine dioxide generationcan be prior to administration by contact of the chlorinedioxide-generating components with the appropriate agent (e.g., aqueousmedium, electromagnetic energy, etc). Alternatively, chlorine dioxidegeneration initiated in situ, by contact with an aqueous medium, such asmucus, saliva, water, wound exudate or the like.

Armed with this disclosure and the knowledge in the art, it is withinthe skill of the skilled artisan to physically adapt the devices andcompositions that deliver a substantially oxy-chlorine anion freechlorine dioxide composition for contact with a wound to treat thewound, contact with a tooth surface for tooth whitening or contact withoral mucosal tissue such as gingival mucosal tissue to treat an oralcavity infection. For instance, the devices and compositions for use inoral applications can themselves be in the form of a dental tray or canbe adapted to be delivered by means of a dental tray.

3. Treatment Regimens

In an embodiment, the method can comprise a single administration of thecomposition comprising a chlorine dioxide source. In another embodiment,the method comprises one or more iterations of the contacting step,wherein each subsequent administration step is substantially contiguousto the previous iteration. In some embodiments, the method comprisesthree, four, five, or more substantially contiguous iterations of anadministration step. Each iteration can be identical in terms of doseand contact duration, each iteration can be different, or a combinationof both. As shown herein, multiple substantially contiguous applicationsto a tissue of a composition comprising a chlorine dioxide source can beefficacious in reducing bacterial count in a wound. As further shownherein, multiple substantially contiguous applications of compositioncomprising a chlorine dioxide source is efficacious in tooth whitening.Specifically, it was found that for a given total dosage (Concentrationof chlorine dioxide in parts-per-million×total time of exposure in min;ppm-min), statistically significantly improved tooth whitening can beachieved by using more frequent, shorter duration applications in asubstantially contiguous manner. Each application uses a fresh specimenof the composition comprising a chlorine dioxide source. By “freshspecimen” is meant a specimen of composition that has not beenpreviously exposed to a biological tissue. Accordingly, a fresh specimenhas undergone minimal-to-no chlorine dioxide decay. In exemplaryembodiments, the composition for a treatment is freshly made. As usedherein, “freshly made” means that the addition of chlorine dioxide tothe other components of the final composition occurs within about onehour, within about 30 minutes, or within about 15 minutes beforecontacting a tissue with the composition. A freshly made composition hastherefore undergone minimal-to-no chlorine dioxide decay.

In the iterative embodiments, while the composition comprising achlorine dioxide source can be the same in the iterations, it is morecommon that the composition in each iteration is fresh. In other words,the composition in one iteration is replaced with a fresh specimen ofthe composition. In embodiments using devices or compositions to delivera substantially oxy-chlorine anion free chlorine dioxide composition toa tissue, the device or composition in one iteration is replaced with afresh device or specimen of composition. By “fresh device” is meant adelivery device whose tissue interface for delivering ClO₂ has not beenpreviously exposed to a biological tissue. Accordingly, a fresh devicehas undergone minimal-to-no chlorine dioxide decay. In some embodiments,a single batch of the composition comprising a chlorine dioxide sourceis prepared at the start of treatment in a volume sufficient to coverthe entire series of contiguous iterations, and fresh specimens aretaken from the single batch for each iteration. In other embodiments,the composition comprising a chlorine dioxide is prepared fresh beforeeach iteration.

In other embodiments, the method can further comprise alternatingtreatment steps wherein one step comprises administration of acomposition comprising a chlorine dioxide source and a second stepcomprises administration of a composition comprising a second,non-chlorine-dioxide therapeutic agent. These steps may take place inany order and in multiple iterations. Consecutive steps may comprise thesame composition or different compositions. Examples of othernon-chlorine dioxide therapeutic agents are listed elsewhere herein.

The method can comprise two or more sequential steps of administrationof the composition comprising a chlorine dioxide source, followed by atleast one step of administration of the other therapeutic agent. Thenumber and/and duration of administration steps with the compositioncomprising a chlorine dioxide source can be the same or different as thenumber and/or duration of administrations with the second therapeuticcomposition. The composition comprising a chlorine dioxide source can beidentical in the plural steps or can be different, such as a differentconcentration of chlorine dioxide. Similarly, the second therapeuticagent composition can be identical in the plural steps or may bedifferent. Likewise, the duration of treatment steps can be the same ordifferent for the composition comprising a chlorine dioxide source andfor the second therapeutic agent composition.

Treatment can occur as frequently as several times daily, or can occurless frequently, such as once, once a day, once a week, once every twoweeks, once a month, or even less frequently, such as once every severalmonths or even once a year or less. The frequency of treatment suitablefor achieving the desired efficacy will be readily apparent to theskilled artisan and will depend upon any number of factors, such as, butnot limited to, the type and severity of the disease/disorder beingtreated and the method of contacting the tissue, etc. As discussedelsewhere herein, a treatment can comprise one episode of tissue contactor more than one episode. Treatment episodes can be: substantiallycontiguous, separated in time (e.g., a few hours to a few days, a fewdays to a few weeks, and also longer intervals including several monthsto a year or more) or both. In some embodiments, treatment comprises atleast two substantially contiguous episodes of tissue contact. Thecontiguous episodes can be the same duration in time such as about 7.5minutes or different durations of time such as 5 minutes and 10 minutes.In some embodiments for whitening a tooth surface, treatment is at leastfour substantially contiguous episodes of 7.5 minutes each. In oneaspect of this embodiment, the composition comprises between about 100to 400 ppm ClO₂, or about 150 to about 200 ppm ClO₂. The pH of thecomposition in these embodiments can be about 4 to 7, or about 5 to 6.

In an embodiment for treating wounds, a combination therapy includingdebridement is envisioned. Debridement is the process of removingdamaged tissue, necrotic tissue and/or infected tissue in a wound.Debridement is believed to be benefit wound healing by improving thehealing potential of the remaining healthy tissue. In general,debridement can either be done surgically, mechanically, chemically,and/or with maggot therapy, and these procedures are well-known in theart. Debridement can also be achieved by use of ultrasonic energy. Inthis case, the ultrasound treatment would precede the step of contactingthe wound with a chlorine dioxide composition.

In an embodiment, a combination therapy including high frequencymechanical energy to improve contact of the chlorine dioxide with thetarget tissue is envisioned. An exemplary frequency range is about 5 Hzto about 5 MHz.

In one embodiment, the high frequency mechanical energy is ultrasound.Ultrasound is sound energy of frequency >20 kHz to about 10 MHz, whichis above the normal range of human perception, and with power fromgreater than 0 to about 5 W/cm². It may produce a number of biophysicaleffects that are relevant to wound healing. These include alterations incellular protein synthesis and release, blood flow and vascularpermeability, angiogenesis, and collagen content and alignment. Woundand tissue care methods using ultrasonic energy in wound care requiresultrasonic energy to be emitted from a radiation surface to the wound ortissue surface. In one embodiment, a tissue to which a compositioncomprising a chlorine dioxide source has been applied is then exposed toultrasonic energy to increase penetration of the chlorine dioxide intothe tissue. Increasing tissue penetration is envisioned to improve woundhealing by increasing contact of chlorine dioxide with pathogens locatedbelow the surface of the wound. In certain embodiments, reduction inbacterial count in a combination therapy including ultrasonication (incomparison to the same therapy in the absence of the ultrasonication)can be from at least about 1 log, 2, logs, 3 logs, 4 logs, up to asubstantially complete log kill (e.g., substantially no bacteriadetectable). For instance, for a wound having an initial total bacterialcount of 8 log prior to treatment, a substantially complete log kill is8 logs. Bacterial count can be assessed by any method known in the art.An exemplary method for assessing bacterial count is by obtaining atissue sample of the infected tissue, preparing serial dilutions fromthe tissue sample, and plating the dilutions to culture and assessbacterial count. Assessment before and after treatment is performed toassess the reduction in bacterial log.

In some embodiments, the ultrasound frequency can be from about 1 kHz toabout 100 kHz, about 10 kHz to about 50 kHz, or about 20 kHz to about 40kHz and all integer values therebetween. For instance, in certainembodiments using ultrasonic energy, the frequency ranges from 20 kHz,21, 22, 23, 24, 25 to 40 kHz, from 26 kHz, 27, 28, 29, 30, to 40 kHz,from 31, 32, 33, 34, 35, to 40 kHz, or from 36 kHz, 37, 38, 39 to 40kHz. In some embodiments, at least two different ultrasonic wavefrequencies administered intermittently can be used. In someembodiments, power intensities can range from about 0.1 to about 5W/cm², including all one-tenth values therebetween. Therefore, powerintensities can be from about 0.1, 0.2, 0.3, 0.4, 0.5 to about 5 W/cm²from 0.6, 0.7, 0.8, 0.9, 1.0 to about 4.5 W/cm², from 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7. 1.8, 1.9 to 4.5 W/cm², from 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7. 2.8, 2.9 to 4.5 W/cm², from 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7. 3.8, 3.9 to 4.4 W/cm², or from 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7. 3.8, 3.9 to 4.0 W/cm². Duty cycle can range from about 20% toabout 100%.

In some embodiments, at least two different power intensitiesadministered intermittently can be used. Duty cycles can be the same ordifferent. The duration of exposure to ultrasonic energy can be the sameor different as the duration of exposure to the composition comprising achlorine dioxide source, In some embodiments, the duration of ultrasonicenergy for a single episode ranges from about 10 seconds to about 10minutes, from about 10 seconds to about 5 minutes and from about 30seconds to about 5 minutes, and all ranges inbetween. During a giventreatment, multiple episodes of ultrasonic energy administration can beadministered. The multiple episodes can be substantially contiguous orspaced out, for instance to permit cooling of tissue temperature betweenadministrations of ultrasonic energy. In one embodiment, a tissuecontacted with a composition comprising chlorine dioxide comprisingabout 100 to about 400 ppm chlorine dioxide is exposed to ultrasonicenergy for between about 2-4 minutes using a frequency of about 20 to 40kHz, with a power intensity from about 2 to about 4 W/cm² or from about2.5 to about 3.5 W/cm², and a 40% duty cycle. Optionally, a tissue iscontacted with at least two, three, four or more iterations of thisultrasound treatment.

Ultrasonication is also envisioned to improve alleviation of an oraltissue infection by improving penetration of chlorine dioxide intosupra- and sub-gingival pockets. Ultrasound devices for oral applicationare known in the art. See, for instance, U.S. patent publication no.20080209650 and U.S. Pat. Nos. 6,881,061 and 7,044,737.

In another embodiment, the method can comprise a combination therapysuch as the addition of a second therapeutic agent. Combination therapyrefers to the addition of another step intended to improve woundtreatment or alleviation of oral tissue infection. In one embodiment, acombination therapy of contacting tissue with a chlorine dioxidecomposition and another API is envisioned. In one embodiment, the API isan antimicrobial, another therapeutic agent, or a combination thereof.Exemplary antimicrobials are disclosed elsewhere herein. Othertherapeutic agents include topical anesthetics, steroids, analgesics andthe like. Optionally, the method can further comprise the step ofapplying ultrasonic energy to the chlorine dioxide composition that hasbeen contacted to the wound.

In another embodiment, a combination therapy including a pre-treatmentis envisioned. The step of pre-treatment of the tissue is intended toincrease penetration of the chlorine dioxide into the tissue. Anexemplary pre-treatment includes, but is not limited to, contacting thetissue targeted for treatment with dimethylsulfoxide (DMSO) following byadministering a composition comprising a chlorine dioxide source. Otherexemplary pre-treatments include treating tissue with electromagneticsource following by administering a composition comprising a chlorinedioxide source, treating tissue with fatty acids following byadministering a composition comprising a chlorine dioxide source, ortreatment with sucrose esters, following by administering a compositioncomprising a chlorine dioxide source. See, e.g., U.S. Publication No.20080208179 and U.S. Pat. No. 4,865,848.

4. Tissue Irrigation

In another embodiment, the methods of administering or contacting atissue with a composition comprising a chlorine dioxide source can bepracticed using irrigation. Irrigation of a tissue, such as a wound oran oral cavity infection, refers to the process of rinsing the tissuewith a fluid composition. Irrigation can be either continuous orintermittent. Intermittent irrigation can be practiced using anautomated irrigation device modified to deliver periodic applications ofthe composition to a tissue or can be practiced by manual irrigation,such as rinsing the wound or oral cavity with a volume of a compositioncomprising a chlorine dioxide source, and repeating this stepiteratively using a fresh volume of the composition in each iteration.Continuous irrigation has the advantage that the effective concentrationof chlorine dioxide per square inch of compromised skin is maintained ata substantially constant level over time and can be kept at an excesssuch that an efficacious amount is delivered, even though some chlorinedioxide may be consumed initially by reacting with organic matter, suchas protein in wound exudate. As such, irrigation of a wound is expectedto be equally effective or more effective than existing state-of-the-artproducts for wound treatment, e.g., silver-containing plaster or gels.Irrigation of infected oral tissue is expected to improve contact withsupra- and sub-gingival pockets, which can improve reduction inbacterial count.

An irrigation system for use in the methods described herein isgenerally comprised of two components: a) an irrigation devicecomprising a chamber with inlet and outlet ports which covers the woundor other tissue to be irrigated; and a means of supplying solution intoand draining solution out from the chamber; and b) a fluid compositioncomprising a chlorine dioxide source, which is supplied to the chamberso as to treat the wound or topical lesion. More specifically, theirrigation device can have five components: 1) a flexible, semi-rigid orrigid pouch or other containment chamber with inlet and outlet ports andwith an opening that contacts at least a portion of tissue targeted tobe irrigated; 2) a fluid supply and egress system which can be connectedto the chamber ports so as to provide fluid to or drain fluid from thechamber; 3) a means of maintaining contact of the chamber to the tissuewhich surrounds the target tissue so as to form a tight substantiallyleak-proof seal; 4) an optional open cell foam or other porous materialthat can be placed inside the pouch to ensure a uniform distribution offlow; and 5) a fluid handling unit which supplies fluid to and/or allowsfluid to exit the containment chamber.

The first component of the proposed system is the pouch or containmentchamber. In exemplary embodiments, the outer surface of the pouch ismade from one or more semi-rigid or rigid materials when suction is tobe applied or from one or more soft flexible materials when thecontainment vessel is always under positive pressure. Thethree-dimensional shape of the chamber can assume multipleconfigurations. In one embodiment, the open portion of the chamberconforms to or is slightly larger than the boundary of the tissue thatis being treated, and that the volume of the pouch is sufficient toprovide an appropriate turnover of irrigation fluid. Another optionalfeature of the pouch is that it can be constructed of materials that areeither antimicrobial or actively prevent biofilm formation.

The second component of this system is the system which allows fluid toflow into and out of the containment chamber. The system can be assimple as a single lumen device which only supports irrigation. However,in an embodiment, the tubing contains two or more lumens, one of whichcan be used to supply other agents such as a non-chlorine dioxideantimicrobial agent or an anesthetic agent to the pouch for use intissue therapy, and one of which can be used to apply vacuum to thetissue area and/or can be used for suction.

The tubing system in an embodiment is a single or dual line flexibletube which is fabricated from flexible PVC or from a silicone basedmaterial. However, those skilled in the art will recognize that anysuitable material can be used to fabricate the tubing, and will alsorecognize that the number of tubes chosen can vary, so long as theyachieve the goals of the methods, devices and compositions disclosed.For example, one lumen can be used to apply a vacuum, and another lumenmay be used to supply antibiotics, to irrigate a wound, or even toprovide gases (e.g., oxygen) which may assist, for instance, in healingthe wound. In addition, a third lumen can be used to conduct anelectrical line which provides electrical stimulation. The lumens can beseparated from one another, adjacent to one another, concentric to oneanother, or any combination thereof.

The third system component is a means of maintaining the chamber incontact with the skin so as to provide a tight, substantially leak-proofseal. The system can be relatively simple, such as a strap wrappedaround the body in the location of the target tissue such that the strapholds a rigid chamber in place over the target tissue. In an embodiment,the attachment system is comprised of two components: 1) an adhesivewafer formed from a polyurethane film to which is laminated a suitable,skin-friendly adhesive, for example an acrylic adhesive; and 2) anannular wafer of similar construction wherein the urethane film iswelded to the pouch material. The chamber is attached to the skin byfirst cutting a hole in the adhesive wafer which conforms to the outlineof the wound or other topical lesion, and then attaching that wafer tothe skin; the annular wafer attached to the pouch is then attached tothe urethane film on the “skin side” adhesive wafer.

The fourth component is the flow distribution component which,optionally, is placed inside the irrigation chamber to ensure an evendistribution of flow. This component can be a perforated membrane, anarray of perforated tubes, or any other device known in the art forimproving the uniformity of fluid flow. In an embodiment, the preferredflow distribution component is a porous open cell foam located in theopening of the chamber and in contact with the target tissue.

The fifth component is the fluid handling unit which supplies fluid toenter and/or allows fluid to exit the containment chamber. The fluidhandling unit can be of any design which allows for the flow of fluidinto or out of the chamber. For example, a fluid supply vessel can belocated in a position of higher elevation relative to the chamber and beconnected to the chamber by tubing such that fluid can flow into thechamber from the vessel by gravity. Similarly, a used-fluid receivingvessel can be located at an elevation lower than the chamber and beconnected to the chamber by tubing such that used-fluid may flow out ofthe chamber into the fluid receiving vessel by gravity. In anembodiment, this component is a portable, lightweight, battery operatedpump which attaches to the proximal end of at least one lumen of thetubing. Of course, other lumens can be attached to other items, such asantibiotic drip devices, electrical devices, etc.

In an embodiment, the fluid handling unit is designed such that it canbe easily carried by a shoulder attachment or by attachment to the beltof the patient. In addition to the vacuum pump, in exemplaryembodiments, the drain/suction unit also includes a reservoir, a batterypower supply, and control switches for turning the drain/suction unit onor off.

Since chlorine dioxide can be lost to consumption by contaminants ordiffusion through a material of some part of the irrigation device, thechlorine dioxide concentration within the containment chamber can belower than that which exists in the fluid supply vessel containing thefresh composition comprising chlorine dioxide. It is important, then, toprovide a sufficient flow and concentration of fresh chlorine dioxidemixture to the chamber to maintain the chlorine dioxide concentrationwithin the chamber at a desired efficacious level. The chlorine dioxideconcentration of the incoming fluid mixture should be at least as highas the desired concentration within the chamber. In certain embodiments,the incoming fluid mixture should be at a higher concentration than thetarget concentration. If the concentration of chlorine dioxide in thechamber is higher than the desired concentration, then the rate of flowinto the enclosure may be reduced or the concentration of the incomingsolution can be decreased, or both changes can be made until thechlorine dioxide concentration in the chamber falls to the desiredlevel. If the concentration of chlorine dioxide in the chamber is lowerthan the desired concentration, then the rate of flow into the enclosurecan be increased or the concentration of the incoming solution may beincreased, or both changes can be made until the chlorine dioxideconcentration in the chamber rises to the desired level.

The irrigation device described can be used with a substantiallynon-cytotoxic and/or substantially non-irritating composition comprisinga chlorine dioxide source to contact the tissue with a continuous streamof the composition. In another embodiment, the irrigation device can bemodified by the addition of an oxy-chlorine anion barrier. Specifically,the device contemplated herein comprises a chamber comprising anoxy-chlorine anion barrier, wherein the device has an inlet port forsupplying a chlorine dioxide solution into the chamber and an outletport for removing chlorine dioxide solution and an opening covered bythe oxy-chlorine anion barrier. The chamber is designed to form a tightsubstantially leak-proof seal with the tissue surrounding an infectedarea, wherein the opening is proximal to the infected area. Theoxy-chlorine anion barrier is interposed between the infected area andthe chamber opening. The chlorine dioxide solution containingoxy-chlorine anions is introduced into the chamber, and chlorine dioxidepasses through the oxy-chlorine anion barrier covering the opening andthereby contacting the infected area, while the passage of oxy-chlorineanions through the barrier is limited to substantially non-cytotoxicand/or substantially non-irritating levels. Exemplary materials usefulas an oxy-chlorine anion barrier are described elsewhere herein.

5. Dose and Duration

The amount of the chlorine dioxide delivered by the compositioncomprising a chorine dioxide source varies within wide limits and can beadjusted to the individual requirements in each particular case. Theamount depends on the condition treated, the general state of health ofthe recipient, the number and frequency of administrations and othervariables known to those of skill in the art. Accordingly, the amount ofchlorine dioxide to be delivered to a tissue (i.e., an efficaciousamount) will relate to the result intended from the application ofchlorine dioxide to the tissue. The skilled artisan can readilydetermine the appropriate amount or amount range of chlorine dioxide tobe efficacious for a given use. Generally, useful amounts comprise, forexample, from about 1 to about 2000 ppm chlorine dioxide, at least about1 to about 1000 ppm or at least about 20 to about 400 ppm. In someembodiments, the chlorine dioxide is present in the composition in atleast about 5 ppm, at least about 20 ppm, or at least about 30 ppm.Typically, the amount of chlorine dioxide can range to about 1000 ppm,up to about 700 ppm, up to about 500 ppm and up to about 200 ppm. In oneembodiment, the composition comprises about 30 to about 100 ppm chlorinedioxide. In some embodiments, a useful dose range can be from about 2.5mg chlorine dioxide per area of contact (in square meters) to about 500mg/m² chlorine dioxide. Doses of at least about 10 mg/m², at least about15 mg/m² and at least about 20 mg/m² can also be useful.

The duration of contact with the tissue to obtain efficacy can bereadily determined by the skilled artisan in view of the teachingsherein and the knowledge in the art. The duration of contact will beinfluenced, for instance, by the type of infection, the presence orabsence of biofilm, the tissue type, whether treatment is therapeutic orprophylactic, and the formulation of the chlorine dioxide composition(e.g, liquid or gel or a slow-release formulation). Advantageously, evenafter prolonged contact, the composition does not substantially irritatemucosal or dermal tissue. Similarly, substantially contiguous iterationsof contact with fresh specimens do not substantially irritate mucosal ordermal tissue. Generally, duration of contact ranges from seconds tominutes to hours to days. In some embodiments, the duration of contactcan be at least about 60 seconds, at least about 1, 2, 3, 4, or 5minutes, at least about 6, 7, 8, 9, or 10 minutes, or at least about 11,12, 13, 14, or 15 minutes. In some embodiments, contact duration canrange up to 16, 17, 18, 19, or 20 minutes, further up to 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 minutes, and further up to about 35, 40, 45,50, 55, or 60 minutes or longer in some circumstances. In certainembodiments, duration of contact ranges between about 1 and about 60minutes, from about 5 minutes to about 30 minutes, or from about 10 toabout 20 minutes. In some embodiments, duration of contact for atreatment is about 15 minutes. In some embodiments, the duration ofcontact ranges from at least about one (1) hour to about 72 hours, fromat least about 8 hours to about 48 hours, or from at least about 12hours to about 36 hours. In certain embodiments, duration of contactranges from about 1 hour to about 6 hours, or from about 1.5 hours toabout 4 hours.

Dosage of chlorine dioxide can be expressed in terms of concentration ofchlorine dioxide (in parts-per-million) times the duration (in minutes)of tissue contact with the chlorine dioxide, with the unit of ppm-min.Dosage refers to a single treatment. The concentration of chlorinedioxide is with respect to the composition used in the single treatment.The single treatment can comprise a single contacting step or multiplecontacting steps (e.g., substantially contiguous iterations). In someembodiments, dosage in terms of ppm-minute can range from about 100ppm-minutes to about 10,000 ppm-minutes, or from about 200 ppm-minutesto about 5000 ppm-minutes. In embodiments where the method is practicedon an infection comprising a biofilm, dosage of at least about 200ppm-minutes is useful. These ranges are appropriate for applications in“clean” systems, e.g., application having little or no organic materialother than the pathogens. In embodiments for treating infectedbiological tissue comprising a protein-rich environment, such as woundexudate, these ranges are suitable target ranges for the actual dosageadministered, as discussed below, to achieve a significant reduction inbacteria or other pathogens.

The amount of chlorine dioxide in a composition can decrease due to thepresence of organic material, such as protein. For instance, wound bedsare protein-rich environments that comprise blood serum. Actual dosageof chlorine dioxide therefore varies depending on the amount of woundfluid, the percent blood serum in the wound fluid and the amount ofchlorine dioxide in the treatment composition. An actual dosage istherefore the dosage corrected for the loss of chlorine dioxide due tothe presence of blood serum in a wound bed. As shown herein, one canestimate the actual chlorine dioxide concentration in a woundenvironment as a function of time, and thereby estimate an actual dosageduring a chlorine dioxide treatment in the presence of wound fluid. Theactual dosage estimate is calculated by determining the ClO₂concentration in solution as a function of time, and then integratingthat equation over the contact time of treatment. The details are asfollows.

First, one calculates the starting weight ratio of blood serum to ClO₂(BS/ClO₂). To do that, one has to estimate the estimate milligrams ofblood serum BS in the wound by estimating the weight of wound fluid WFin the wound (in mg). It is assumed that wound fluid generally containsabout 5% by weight blood serum. Therefore, to estimate the milligrams ofblood serum BS in the wound, WF (in mg) is multiplied by 5%:BS (in mg)=WF (in mg)×5%  (1)

To calculate the weight of ClO₂, one must know the amount in mg oftreatment composition Mt that will be applied to the wound, and the ppmClO₂ in that treatment material. The mass of ClO₂ in the treatmentcomposition is then calculated using the equation below:mg ClO₂=Mt (mg)×[ClO₂]/1,000,000  (2)where: Mt=mass of the treatment composition in mg, and [ClO₂]=ClO₂concentration in ppm in the treatment composition. The mg of blood serumin the wound is divided by the mg of ClO₂ in the treatment compositionto obtain the BS/ClO₂ ratio:BS/ClO₂=BS (in mg)/mg ClO₂  (3)

As shown herein, the decay rate K2 of chlorine dioxide as a function ofthe ratio of the blood serum to the mass of ClO₂ in the treatmentcomposition is estimated by the equation:K2=−0.00927(BS/ClO₂)+1  (4)Therefore, K2 is estimated by plugging in the result of equation 3 intoequation 4. K2 is then plugged into the equation:Ct=Co×t^K2  (5)where Ct=the concentration of ClO₂ as a function of time; Co=the initialClO₂ concentration in ppm; and t=time in minutes. This function is thenintegrated as a function of time over the treatment time of the wound.The result of the integration is an estimate of the actual dosage in thewound. This estimate and the minimum efficacious dose in a clean system(discussed above) can enable a clinician to chose an appropriatetreatment composition regarding the chlorine dioxide concentration inthe treatment composition. In some embodiments, the method comprisestreatment of an actual dosage of about 200 ppm-min to about 15,000ppm-min, about 500 ppm-min to about 5000 ppm-min, or about 750 ppm-minto about 2000 ppm-min.

EXAMPLES

The compositions and methods of use are further described in detail byreference to the following experimental examples. These examples areprovided for purposes of illustration only, and are not intended to belimiting unless otherwise specified. Thus, the compositions, methods ofuse, and systems should in no way be construed as being limited to thefollowing examples, but rather, should be construed to encompass any andall variations which become evident as a result of the teaching providedherein.

Example 1 Chlorine Dioxide Efficacy Against Biofilm

Chlorine dioxide treatment involves exposing micro-organisms to chlorinedioxide for a period of time. Treatment conditions, often called thedosage of chlorine dioxide, can be determined by calculating theintegral of the chlorine dioxide concentration as a function of time,C(t), over the treatment time of use:Dose=∫₀ ^(T) C(t)dt  (6)

In systems where the chlorine dioxide concentration is generallyconstant over the treatment time (e.g., varies by less than ±10%relative) or varies linearly over the treatment time, an averagechlorine dioxide concentration can be calculated by averaging thestarting and ending concentrations and then multiplying that averagechlorine dioxide concentration during an exposure period by the time ofexposure (Average Concentration×time):

$\begin{matrix}{\frac{\left( {{Ct} + {Co}} \right)}{2} \times t} & (7)\end{matrix}$where Ct is the ending ClO₂ concentration at time t, and Co is theinitial chlorine dioxide concentration. Dosage is commonly given inunits of ppm-minutes.

A series of experiments were conducted to estimate the dosage ofchlorine dioxide needed to kill bacterial biofilms consisting ofPseudomonas aeruginosa (PA), methicillin resistant Staphylococcus aureus(MRSA), or a combination of both.

Bacterial biofilms were grown for between 7 and 10 days on non-porousceramic disks in a spinning disk biofilm reactor. The reactors wereinoculated with PA, MRSA, or a mixture of MRSA and PA bacteria toproduce PA, MRSA, or mixed PA/MRSA biofilms respectively.

Chlorine dioxide solutions were produced at different nominalconcentrations using ASEPTROL® S-Tab10 tablets, and the exactconcentrations were then measured using a Hach Model 2400 UV/Visspectrophotometer in accordance with the manufacturer's instructions.Pairs of biofilm-containing disks were immersed for different times insolutions of different chlorine dioxide concentrations, neutralized withdilute sodium thiosulfate solution, and then plated to determine thenumber of surviving bacteria on each disk. Two untreated disks were alsoexposed to neutralizing solution and plated to determine the startingbacterial counts. In all tests, the baseline bacterial counts were inthe range of 10⁷ to 10^(7.5) colony-forming units per disk (cfu/disk). Aone log reduction refers to reducing the cfu/disk by one order ofmagnitude, e.g., a reduction from 10^(7.1) cfu/disk to 10^(6.1)cfu/disk. Accordingly, a substantially complete kill is achieved in theexample if the log reduction of bacteria is about 7 to 7.5.

Table 1 summarizes the test conditions used in the differentexperiments. The results are presented in terms of log reduction of thedifferent bacterial organisms. In the case of mixed PA/MRSA biofilms,results are given as total bacteria. The data are also shown in FIG. 1.

TABLE 1 Log ClO2 Reduction Exposure Concen- Conc × in Time tration TimeOrganism Sample # Organism (minutes) (ppm) (ppm-min) Count 1 MRSA 1.0208 208 5.5 2 MRSA 5.0 204 1020 7.1 3 MRSA 10.0 45 450 5.5 3 MRSA 30.040 1200 5.1 4 MRSA 30.0 175 5250 7.1 6 PA 0.5 200 100 5.9 7 PA 5.0 43215 5.7 8 PA 5.0 187 935 7.5 9 PA 10.0 37 370 7.5 10 PA 50.0 35 1750 7.511 PA 50.0 166 8300 7.5 12 MRSA + PA 10.0 52 520 5.5 13 MRSA + PA 30.052 1560 7.4 14 MRSA + PA 1.0 181 181 5.1 15 MRSA + PA 5.0 160 800 7.4 16MRSA + PA 30.0 160 4800 7.4

These data show that complete kill of either a PA biofilm, a MRSAbiofilm or a mixed PA/MRSA biofilm was achieved with a dosage of betweenabout 400 and 1000 ppm-minutes (see Samples 2, 9 and 15). A PA biofilmappeared slightly easier to kill than a MRSA biofilm, but the differenceis small. A kill of about 5 orders of magnitude (i.e., 5 log) can beachieved at a dosage of greater than about 200 ppm-min (see Samples 1,7, and 12). These data demonstrate that chlorine dioxide has greatpotency for disrupting and eradicating biofilms using readily achievabledosages, which are substantially non-cytotoxic and/or non-irritating tobiological tissue.

Example 2 Chlorine Dioxide Loss in Protein-Rich Solution

Oxidizing biocides can react with and be destroyed by organic material,such as proteins. While chlorine dioxide is generally recognized as arelative unreactive oxidizing biocide, chlorine dioxide can be consumedin the organic-rich treatment environment of a wound. Blood serum is aprotein-rich material and is a particularly reactive component ofwounds.

A series of experiments were conducted to estimate and quantify theeffect of blood serum on chlorine dioxide concentration. Solutions ofchlorine dioxide and fetal blood serum (FBS) were prepared at differentconcentrations of chlorine dioxide and FBS, and the chlorine dioxideconcentration was tracked using a Spectral Physics UV/Visiblespectrophotometer with a direct insertion probe (1 mm path length).

The initial chlorine dioxide concentration was measured using a HachModel 2400 UV/Vis spectrophotometer. A UV/Vis scan was then measuredover the wavelength range of about 200 to 600 nm using the directinsertion probe, and this was used to calculate the initial absorbanceof the solution. FBS was then added, and UV/Vis scans were runperiodically thereafter. The area of the chlorine dioxide absorbancepeak was calculated for each scan, and the relative chlorine dioxideconcentration at each time was calculated by dividing the area at eachtime point by the peak area of the initial scan (prior to FBS addition).

Relative chlorine dioxide concentration was plotted versus time for eachset of chlorine dioxide and FBS concentrations. The curves were well fitby regression to a function of the form:Ct/Co=t^(K2)  (8)

where Ct is the ClO₂ concentration at time t, and Co is the initialchlorine dioxide concentration. Data for a solution containing 6.29 wt.% FBS and having an initial FBS/ClO₂ weight ratio of nominally 500(mg/mg) are depicted in FIG. 2. The exponent K2 in the equation is anestimate of the decay rate of chlorine dioxide in the FBS solution. Theweight ratio of FBS to chlorine dioxide was calculated by dividing themass of FBS added to the solution by the mass of chlorine dioxide in thesolution.

The decay rates for the different solution concentrations were thenplotted versus the initial mass ratio of FBS/ClO₂ for each system. Theseresults are depicted in FIG. 3. FIG. 3 shows that the decay rate K2decreases linearly with the initial FBS/ClO₂ ratio, and fits theequation:K2=−0.00927(FBS/ClO₂)+1  (9)

Using this relationship, it is possible to estimate the actual chlorinedioxide concentration in a wound environment as a function of time, andthereby estimate an actual dose during chlorine dioxide treatment in thepresence of wound fluid. By targeting an actual dose of greater thanabout 500 ppm-min, one can expect nearly complete kill of accessiblebiofilm in the wound. An actual dose of greater than about 200 ppm-minshould yield greater than about 5 log kill of accessible biofilm.

Example 3 Chlorine Dioxide Efficacy Against Biofilm in Protein-RichEnvironment

A series of experiments were conducted to determine the effect of FBS onthe kill of PA, MRSA and mixture PA/MRSA biofilms. Biofilms were grownas described in Example 1. Disks were immersed in 25 ml of FBS solutionat double the target FBS concentration, and to that was added 25 ml ofchlorine dioxide solution at double the target chlorine dioxideconcentration. After the desired contact time, disks were removed fromthe treatment solutions, neutralized, and plated as described above.

Treatment conditions and results are shown in Table 2. Dosage is givenboth as the nominal dose based upon the initial chlorine dioxideconcentration, and the actual dosage as estimated from the initialchlorine dioxide concentration and the decay relationships describedabove.

TABLE 2 5 9 10 11 3 4 Nominal 6 8 Final Average Corrected 1 ExposureInitial ClO₂ Dosage, Log Reduction 7 Decay ClO₂ ClO₂ Dosage, Sample 2Time Concentration Conc × Time in Organism mg FBS/ rate, Concentration,Concentration, C × t, # Organism (minutes) (ppm) (ppm-min) Count mg ClO₂K2 ppm ppm ppm-min 17 MRSA + PA 15.0 191 2865 8.2 131 −0.2134 107 1492236 18 MRSA + PA 40.0 191 7640 8.2 131 −0.2134 87 139 5559 19 MRSA + PA15.0 240 3600 8.2 104 0.0344 240 240 3600 20 MRSA + PA 30.0 240 7200 8.2104 0.0344 240 240 7200 21 MRSA + PA 60.0 240 14400 8.2 104 0.0344 240240 14400 22 MRSA 15.0 191 2865 4.7 131 −0.2134 107 149 2236 23 MRSA40.0 191 7640 4.7 131 −0.2134 87 139 5559 24 MRSA 15.0 240 3600 4.7 1040.0344 240 240 3600 25 MRSA 30.0 240 7200 4.7 104 0.0344 240 240 7200 26MRSA 60.0 240 14400 4.7 104 0.0344 240 240 14400 27 PA 15.0 191 2865 7.1131 −0.2134 107 149 2236 28 PA 40.0 191 7640 7.1 131 −0.2134 87 139 555929 PA 15.0 240 3600 7.1 104 0.0344 240 240 3600 30 PA 30.0 240 7200 7.1104 0.0344 240 240 7200 31 PA 60.0 240 14400 7.1 104 0.0344 240 24014400Columns 3-6 are measured data; columns 7-11 are calculated data. For K2having a positive sign (such as Samples 20-21), it was assumed thatthere was insufficient FBS present to cause a material loss of chlorinedioxide. Therefore, for such samples, it was assumed that chlorinedioxide was unchanged over the time of the experiment

FIG. 4 is a graph of the results. These data demonstrate that, like thebacterial kill results in a “clean system” (free of FBS; see Example 1),bacterial kill in a “dirty system” (e.g., rich in organic matter) can bepredicted from the corrected FBS concentration. Specifically, correcteddose rates of about 2000 ppm-min and higher resulted in complete killfor each type of biofilm tested.

Example 4 Chlorine Dioxide Delivery Methods for Wound Treatment

This study was designed to generate initial data for the assessment ofvarious delivery methods of ClO₂ into infected wounds and to ascertainthe drug's effect on microbial bioburden and wound healing.

The test animal was a pig (n=1). Pigs are commonly used as models forwound healing in part because pig skin shares many characteristics withhuman skin. The porcine model is considered to be an excellent tool forthe evaluation of candidate agents intended for use in human wounds.

The pig was housed in accordance with “Guide for the Care and Use ofLaboratory Animals DHEW” (NIH). The pig was fed fresh porcine diet dailyand water was available ad libitum. The pig was housed in atemperature-controlled animal room, having a 12-hour light/dark cycle.The room was kept clean and free of vermin.

On Day 0, the pig was anesthetized and sixteen (15) full-thicknesswounds (eight per side) were created using a custom designed 2-cmtrephine. Each wound was 2-cm in diameter. An epinephrine solution(1:10,000 dilution) was applied to the wounds on gauze sponges untilhemostasis was complete (approximately 10 minutes). Each wound was theninfected with a bacterial inoculum.

Three different bacteria, Pseudomonas aeruginosa, Fusobacterium sp., andcoagulase-negative Staphylococci (CNS), were cultured and used toprepare inoculums for the wounds. On the morning of surgery, thebacterial cultures were washed with sterile saline and resuspended insaline to a final density of approximately 10⁷ CFU/mL. This bacterialsuspension was used to inoculate each wound.

Wounds were dressed with sterile Telfa gauze, and the infections allowedto mature to establish a bacterial biofilm condition. Telfa dressingswere moistened with saline; the excess saline was removed by squeezingthe Telfa dressings. The dressing was then secured in place withTranspore tape (3M, St. Paul, Minn.). As a secondary dressing, a blueabsorbent pad was used to cover all of the wounds. The absorbent layerof the blue pad was placed against the skin for the first few days. Ifwounds looked too dry, the blue pad was changed with the occlusive sideagainst the skin. A layer of a layer of elastic bandage was wrapped overthe blue pad to prevent movement of the dressings underneath. All wounddressing were changed on Day 1.

On Day 2, wounds were treated with a chlorine dioxide composition usingdifferent delivery methods. In brief, wounds A2-A4 were treated withcontinuous irrigation. Treatment for wounds B2-B4 and C2-C4 wasmultiple, substantially contiguous episodes of contact with asubstantially non-cytotoxic chlorine dioxide gel composition. WoundsD2-D4 were treated with intermittent irrigation using a manualirrigation device. A1, B1 and D1 were controls and were not treated withchlorine dioxide. C1 was manually treated with a chlorine dioxidesolution.

Wounds A2-A4: Each wound was individually covered with an irrigationdevice, each having an inlet port to supply a chlorine dioxide solutionand an outlet port for egress of the chlorine dioxide solution. Theinlet ports were connected by flexible tubing to the outlet of athree-head, adjustable speed pump. The inlets of the pump were connectedto the fluid supply source containing a 400 ppm chlorine dioxidesolution. A second set of flexible tubing was connected to the outletports, which drained into a common waste container. The chlorine dioxidesolution was not re-circulated.

The pump was activated to achieve an initial flow rate of approximately80-82 mL/min through each of the three irrigation chambers. Chlorinedioxide concentrations were measured at the outlets using a UVspectrophotometer. The flow was adjusted as required until the outletconcentrations were no less than 85% of the source concentrations.

Wounds B2-B4: A 400 ppm chlorine dioxide gel comprising 2.83 wt % NaCMCwas applied to each wound in 8 separate, contiguous applications. Eachapplication was about 2.5 ml of the gel. For each application, after thewound was contacted with the gel, the wound was covered and remainedcovered for 12 minutes. The dressing was removed and the gel was rinsedoff the wound using saline (taking ˜3 minutes). Thus, the 8 separateapplications were administered over a period of two hours. A masterbatchof the 400 ppm chlorine dioxide gel was prepared using a concentratedNaCMC base gel and a concentrated freshly-prepared solution of chlorinedioxide (prepared using an ASEPTROL S-Tab2 tablet). The solutions weremixed using syringes connected with Luerlok union. The eight aliquotswere taken from this masterbatch for the separate applications.

Wounds C2-C4: These wounds were treated the same as wounds B2-B4, usinga 400 ppm chlorine dioxide gel comprising 1.5. wt. % HPMC. A masterbatchof the 400 ppm chlorine dioxide gel was prepared as described above forthe NaCMC gel; the eight aliquots were taken from the masterbatch forthe separate applications.

Wounds D2-D4: Each wound was covered with a manual irrigation devicehaving syringes. A masterbatch solution of 400 ppm chlorine dioxide wasprepared at the start of the treatments. For each application, a freshspecimen (20 ml) was removed, mixed for 1 minute, then applied to thewound via syringe and left to stand for 9 minutes. There were a total of12 treatments administered over a period of two hours.

Wound C1: Using a syringe, 50 ml of 400 ppm chlorine dioxide solutionwas administered every 5 minutes. Thus, there were 24-50 ml applicationsover a period of two hours. The solution used to irrigate the wound wascollected after irrigation, but was not reused.

Wounds A1, B1 and D1 were not treated. To keep the wounds moist, eachwound was covered with an occlusive dressing until it was time to biopsythe wound.

After treatment, each wound was biopsied. The number of bacteria in eachbiopsy sample was assessed as follows. Biopsy tissue sample was placedinto a pre-weighed vessel containing phosphate buffered saline, and theweight of tissue determined. The tissue sample was then homogenized andserially diluted. The serial dilutions were Drop-Plated and incubated todetermine the bacterial counts. One set of samples were plated onTryptic Soy Agar (TSA) to determine the total bacterial counts in thebiopsy specimen. Another set of samples was plated on Mannitol SaltsAgar (MSA) to determine the number of staphylococci present in thebiopsy specimen. A third set of samples was plated on PseudomonasIsolation Agar (PIA) to determine the number of P. aeruginosa present inthe biopsy specimen. Bacterial counts are expressed as log₁₀ (CFU/g).

The data for total bacteria count is depicted in FIG. 6. The moistcontrols averaged slightly over 8 log of total bacteria per gram tissue.The treated wounds all averaged about 6 or less log total bacteria. TheHPMC gel data set (wounds X2-X4) contained one zero count sample, whichaccounts for the large error shown for this data in FIG. 6. Since theMSA and PIA plates for this same sample both showed positive microbialcultures, this zero result is likely the result of a sample processingerror. Without this zero data included, the average Log CFUs for thissample was 5.8, in line with the other treatments. The data in FIG. 6demonstrate that for all delivery methods, a total bacteria reduction inthe range of 2-4 logs was achieved (2-3 logs if the zero data for theHPMC data set is excluded).

Data for coagulase-negative Staph are depicted in FIG. 7. The moistcontrols averaged over 7 log of total coagulase-negative Staph per gramtissue. The treated wounds all averaged about 6 or less logcoagulase-negative Staph per gram tissue. The data in FIG. 7 demonstratethat for all delivery methods, a reduction of coagulase-negative Staphin the range of 1-2 logs was achieved.

Data for Pseudomonas are depicted in FIG. 8. The moist controls averagedover 8 log of total Pseudomonas per gram tissue. The treated wounds allaveraged slightly more than 5 log or less log Pseudomonas per gramtissue. The data in FIG. 8 demonstrate that for all delivery methods, areduction of Pseudomonas in the range of 3-5 logs was achieved.

The results of this example indicate that the improved irrigationmethods used in this study and also the application of chlorine dioxidein a gel formulation moderately lowered the levels of Staphylococcusaureus. Chlorine dioxide was shown to be potent against Pseudomonas,with log reductions of 3 or greater. Additionally, using the improveddrug-delivery methods in this study, a 1-2 log reduction in both Staphand total bacterial levels was also achieved.

Example 5 Efficacy of Chlorine Dioxide Composition with Sonication inPig Model

An experiment was performed to assess the efficacy of a combinationtherapy of chlorine dioxide administration and sonication on bacteriallevels in wounds. The animal model was a pig. Sixteen wounds wereintroduced and inoculated with bacteria as described in Example 6. OnDay 2, treatment was administered. There were control wounds that werenot treated with either chlorine dioxide or ultrasound. Test wounds weretreated with ultrasonic energy for 4 minutes, using a frequency of about20 kHz, a 40% duty cycle and a range of power intensities up to 3.5W/cm²; each test wound received a total of three sonication treatments.Test wounds were treated with a 400 ppm chlorine dioxide solutionapplied to each wound in 8 separate, contiguous applications of 15minutes duration each (total of 120 minutes of exposure to 400 ppmchlorine dioxide) and each application used a fresh specimen of thesolution. Each treated wound received a total of three×4 minutesonication over the course of the 2 hours of chlorine dioxide treatment.

Wounds were biopsied after treatment as described in Example 4.Bacterial counts were found to decrease significantly (multiple logreductions) in the test wounds as a function of the power intensity. At3.5 W/cm², a substantially complete log kill was achieved.

Example 6 Prophetic-Chlorine Dioxide Efficacy in Pig Model withSonication

An experiment will be performed to further assess the efficacy of acombination therapy of chlorine dioxide administration and sonication onbacterial levels in wounds as a function of different parameters. Theanimal model will be a pig. Sixteen wounds will be introduced andinoculated with bacteria as described in Example 4. On Day 2, treatmentwill be administered. There will be control wounds that are not treatedwith either chlorine dioxide or ultrasound, and control wounds treatedonly with ultrasound or only the chlorine dioxide composition. Testwounds will be designed to study efficacy as a function of differentparameters, including frequency, power intensity and duty cycle ofultrasonic energy, duration of treatment and formulation of thecomposition comprising chlorine dioxide (e.g., liquid, gel, orslow-release). Wounds will be biopsied after treatment as described inExample 4. Further tests can be pursued using additional test animals,if necessary.

Based on the result of Example 5, the use of ultrasound is expected toimprove chlorine dioxide penetration into the tissue and thereforeimprove the log reduction of bacteria in a statistically significantamount, e.g., by at least about 0.5 log and preferably, by at leastabout 1 or 2 logs, in comparison to the use of chlorine dioxidecomposition alone.

Example 7 Tooth Whitening with Multiple, Contiguous Applications

This study was designed to assess the efficacy on tooth whitening of twodifferent regimens of multiple, contiguous applications of a 200 ppmchlorine dioxide gel.

Five human extracted teeth (molars) were used in this study. Each toothwas sectioned twice: first at the tooth's cemento-enamel junction toremove the root portion of the tooth; then sectioned mesio-distally toseparate the tooth into two, separate buccal and lingual halves. Thebuccal and lingual tooth segments thereby revealed exposed axial andgingival portions or planes of dentin (connected by an axio-pulpal oraxio-gingival lineangle of approximately 90°) in each sectioned toothsegment. The sectioned teeth were then stained in concentrated black tea(cooled to room temperature after brewing) until each tooth accumulatedenough stain to be visually graded as “C4” (but no lighter than “A4”)per the Vita Classic Shade Vision Guide.

After staining, the cut sides of each tooth were sealed with an acrylicpolymer (i.e. acrylic-based nail polish). The tooth was then “split” orfunctionally “divided” into a left and right sides (the tooth was notphysically split). To create “dividing line,” a narrow channel was firstcreated down the center of the tooth (running from the occlusal to thegingival or cervical portion of the tooth) using a narrow tungstencarbide or diamond tip dental bur, thereby creating an approximately 0.5to 1.5-mm deep by 1 to 1.5-mm wide channel on either the buccal orlingual enamel surface of the tooth segment. The channel was etchedcarefully with a 34-40% phosphoric acid dental etching gel preparation(being careful to confine the gel only to the enamel within the preparedchannel). The etching gel was then removed by water rinsing, the surfacewas dried, and a thin, narrow band of enamel-dentin adhesive was thenapplied carefully only to the enamel within the narrow channel. Thematerial was allowed to dry in ambient air for 1 minute then carefullydried with a gentle stream of compressed air for an additional 10seconds. The enamel-dentin adhesive was subsequently cured by exposureto a visible light dental curing unit using actinic blue light at anapproximate peak wavelength of 470 nm for multiple, 20 second curingincrements. In order to fill in the channel and gradually build a “wall”or barrier to separate the left and right half of the lingual surface ofthe tooth, a thin, narrow layer or band of composite resin dentalrestorative material was added in several steps and cured with thedental visible blue-light curing unit.

The wall that was built up on each tooth prevents whitening agentapplied on the left side of the tooth to migrate into the whiteningagent that was applied on the right side of the tooth, and visa versa.In this way, it is possible to compare two different treatment regimenson the same tooth, thereby minimizing the biological diversity amongdifferent teeth that may affect whitening efficacy.

Two treatment regimens were tested, both using a nominal 200 ppmchlorine dioxide gel comprising sodium carboxymethylcellulose (NaCMC).The actual concentration of chlorine dioxide was measured and found tobe 155 ppm; pH of the composition was 5.0. The general treatmentprotocol is as follows. After mixing, the ClO₂ gel was drawn into a 60ml plastic syringe. The 60 ml syringe was used to store the gel duringthe assay, and for dispensing gel into a 10 ml plastic syringe. The gelin the 10 ml syringe was then dispensed directly onto the tooth sectionenamel surfaces as follows. At time zero, about 1 to 1.5 ml gel wasdispensed onto the enamel surface of each tooth segment attached to theglass microscope slide. The thickness of the resulting gel layer wasabout 1.5 to 3.0 mm in depth. After dispensing the gel onto the toothsegments, the glass slide was placed in a plastic zippered bag,containing small strips of wet paper towel within the bag to maintain100% humidity in the bag. The paper towel strips were positioned toeliminate any contact of the plastic bag with the tooth and gelsurfaces.

Upon conclusion of a contact epidose, the glass slide was removed fromthe plastic bag, and the gel was carefully removed with an extra softbristle toothbrush and a gentle stream of running tap water. The toothsegments were then analyzed for shade, and the gel application procedurewas repeated as designed until the experiment was concluded. The testedtooth segments were stored on the glass microscope slide in 100%humidity for later reference observation. Care was taken to keep theteeth hydrated throughout the experiment to avoid undue color artifactresulting from dehydration. The left side of each tooth was administeredfour separate, substantially contiguous applications, each with acontact length of 15 minutes in duration. The right side of each toothwas administered eight separate, substantially contiguous applications,each with a contact length of 7.5 minutes in duratio. Thus, both sidesof the teeth were exposed to the chlorine dioxide composition for atotal of 60 minutes, with a nominal dosage of 12,000 ppm-min.

Each application used a fresh specimen of chlorine dioxide gelcomposition. Tooth whitening was assessed visually using the VitaClassic Shade Vision Guide. Visual assessment by Vita Classical ShadeGuide: Initial baseline shade and subsequent shade change was assessedby direct comparison to a standard Vita shade guide. The Vita shadeguide is arranged in the following order (as recommended by themanufacturer) for value assessment:B1*A1*B2*D2*A2*C1*C2*D4*A3*D3*B3*A3.5*B4*C3*A4*C4, where B1 is thebrightest and C4 is the darkest. The baseline shade for each tooth ionthis example was C4. Data are reported as change in shade value unit(ΔSVU) from baseline.

The data after 30 minutes of total exposure and after 60 minutes totalexposure are in Tables 3 and 4, respectively.

TABLE 3 30 minutes total ClO₂ exposure Left: 2 × 15 minutes Right: 4 ×7.5 minutes ΔSVU ΔSVU Tooth A 10 12 Tooth B 12 12 Tooth C 11 12 Tooth D12 12 Tooth E 12 15 Average 11.4 12.6

TABLE 4 60 minutes total ClO₂ exposure Left: 4 × 15 minutes Right: 8 ×7.5 minutes ΔSVU ΔSVU Tooth A 12 14 Tooth B 12 12 Tooth C 11 12 Tooth D12 15 Tooth E 12 15 Average 11.8 13.6

These data demonstrate that for a given total exposure time, morefrequent administration of substantially contiguous applications offreshly-made chlorine dioxide composition yields a marked improvement intooth whitening. Specifically, administration of a nominal dosage of6000 ppm-min (30 minutes exposure×200 ppm) in four applications improvedtooth whitening by about 10% compared to administration of 6000 ppm-minin two applications. Similarly, administration of a nominal dosage of12,000 ppm-min in eight applications improved tooth whitening by about15% over administration in four applications.

Based on these data, it is expected that administration of chlorinedioxide by continuous irrigation would yield better whitening resultscompared to the results achieved by applying chlorine dioxidecomposition on a tray for the same duration as the continuous treatment.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the compositions, kits, and their methods of use have beendisclosed with reference to specific embodiments, it is apparent thatother embodiments and variations may be devised by others skilled in theart without departing from the true spirit and scope of the describedcompositions, kits and methods of use. The appended claims are intendedto be construed to include all such embodiments and equivalentvariations.

1. A method for treating a wound in a tissue, the method comprisingadministering to the wound a composition comprising a chlorine dioxidesource to provide an efficacious amount of chlorine dioxide to thewound, thereby treating the wound, wherein the administering stepcomprises one of: i) contacting the wound with a composition comprisingthe chlorine dioxide source and oxy-chlorine anions, wherein thecomposition comprises no more than about 0.25 milligrams oxy-chlorineanion per gram composition and is a substantially non-cytotoxiccomposition: ii) contacting the wound with a device comprising thechlorine dioxide source and oxy-chlorine anions, wherein the devicedelivers a substantially oxy-chlorine anion free chlorine dioxidecomposition comprising no more than about 0.25 milligrams oxy-chlorineanion per gram composition to the wound; or iii) contacting the woundwith a composition comprising the chlorine dioxide source andoxy-chlorine anions; and a barrier substance that substantiallyprohibits passage therethrough of the oxy-chlorine anions and permitspassage therethrough of a substantially oxy-chlorine anion free chlorinedioxide composition comprising more than about 0.25 milligramsoxy-chlorine anion per gram composition, thereby delivering thesubstantially oxy-chlorine anion free chlorine dioxide composition tothe wound.
 2. The method of claim 1, further comprising a seconditeration of the contacting step, wherein the second iteration issubstantially contiguous with the first iteration.
 3. The method ofclaim 2, further comprising at least a third iteration of the contactingstep, wherein the third iteration is substantially contiguous with thesecond iteration.
 4. The method of claim 1, wherein the administrationstep comprises i) contacting the wound with the composition comprisingthe chlorine dioxide source and oxy-chlorine anions to form acomposition-contacted wound.
 5. The method of claim 1, wherein theadministration step comprises i) contacting the wound with thecomposition comprising the chlorine dioxide source and oxy-chlorineanions by irrigating the wound with the composition using an irrigationdevice.
 6. The method of claim 1, wherein the administration stepcomprises ii) contacting the wound with the device comprising thechlorine dioxide source and oxy-chlorine anions, wherein the device isan irrigation device that delivers a substantially oxy-chlorine anionfree chlorine dioxide composition comprising no more than about 0.25milligrams oxy-chlorine anion per gram composition to the wound.
 7. Themethod of claim 6, wherein the administration step comprises contactingthe wound with the device comprising the chlorine dioxide source andoxy-chlorine anions to form a device-contacted wound, and furthercomprising applying ultrasonic energy to the device-contacted wound. 8.The method of claim 1 wherein the administration step comprises iii)contacting the wound with the composition comprising the chlorinedioxide source and oxy-chlorine anions; and the barrier substance thatsubstantially prohibits passage therethrough of the oxy-chlorine anionsand permits passage therethrough of a substantially oxy-chlorine anionfree chlorine dioxide composition to form a composition-contacted wound.9. The method of claim 1, wherein the composition comprising thechlorine dioxide source comprises about 1 to about 1000 ppm chlorinedioxide.
 10. The method of claim 1, wherein the composition comprisingthe chlorine dioxide source has a pH from about 4.5 to about
 11. 11. Themethod of claim 1, wherein an actual dosage of at least about 200ppm-minutes of chlorine dioxide is administered.
 12. The method of claim4, wherein the ultrasonic energy frequency is between about 10 kHz toabout 100 kHz with a power intensity of about 2 W/cm² to about 3.5W/cm².
 13. The method of claim 4, further comprising applying ultrasonicenergy to the composition-contacted wound.
 14. The method of claim 8,further comprising applying ultrasonic energy to thecomposition-contacted wound.
 15. The method of claim 4, furthercomprising at least a second administering step, wherein the secondadministering step comprises one of ii) contacting the wound with adevice comprising a chlorine dioxide source and oxy-chlorine anions,wherein the device delivers a substantially oxy-chlorine anion freechlorine dioxide composition comprising no more than about 0.25milligrams oxy-chlorine anion per gram composition to the tissue; oriii) contacting the wound with a composition comprising a chlorinedioxide source and oxy-chlorine anions; and a barrier substance thatsubstantially prohibits passage therethrough of the oxy-chlorine anionsand permits passage therethrough of a substantially oxy-chlorine anionfree chlorine dioxide composition comprising no more than about 0.25milligrams oxy-chlorine anion per gram composition, thereby deliveringthe substantially oxy-chlorine anion free chlorine dioxide compositionto the wound.
 16. The method of claim 6, further comprising at least asecond administering step, wherein the second administering stepcomprises one of i) contacting the wound with a composition comprising achlorine dioxide source and oxy-chlorine anions, wherein the compositioncomprises no more than about 0.25 milligrams oxy-chlorine anion per gramcomposition and is a substantially non-cytotoxic composition; or iii)contacting the wound with a composition comprising a chlorine dioxidesource and oxy-chlorine anions; and a barrier substance thatsubstantially prohibits passage therethrough of the oxy-chlorine anionsand permits passage therethrough of a substantially oxy-chlorine anionfree chlorine dioxide composition comprising no more than about 0.25milligrams oxy-chlorine anion per gram composition, thereby deliveringthe substantially oxy-chlorine anion free chlorine dioxide compositionto the wound.
 17. The method of claim 8, further comprising at least asecond administering step, wherein the second administering stepcomprises one of i) contacting the wound with a composition comprising achlorine dioxide source and oxy-chlorine anions, wherein the compositioncomprises no more than about 0.25 milligrams oxy-chlorine anion per gramcomposition and is a substantially non-cytotoxic composition; or ii)contacting the wound with a device comprising a chlorine dioxide sourceand oxy-chlorine anions, wherein the device delivers a substantiallyoxy-chlorine anion free chlorine dioxide composition comprising no morethan about 0.25 milligrams oxy-chlorine anion per gram composition tothe tissue.